Hannah L Klein, Ph.D.

The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores
Bermejo, Rodrigo; Capra, Thelma; Jossen, Rachel; Colosio, Arianna; Frattini, Camilla; Carotenuto, Walter; Cocito, Andrea; Doksani, Ylli; Klein, Hannah; Gomez-Gonzalez, Belen; Aguilera, Andres; Katou, Yuki; Shirahige, Katsuhiko; Foiani, Marco
2011 Jul 22;146(2):233-246, Cell
Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes
— id: 136488, year: 2011, vol: 146, page: 233, stat: Journal Article,

Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors
Chi, Peter; Kwon, Youngho; Visnapuu, Mari-Liis; Lam, Isabel; Santa Maria, Sergio R; Zheng, Xiuzhong; Epshtein, Anastasiya; Greene, Eric C; Sung, Patrick; Klein, Hannah L
2011 Aug;39(15):6511-6522, Nucleic acids research
The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair
— id: 136937, year: 2011, vol: 39, page: 6511, stat: Journal Article,

The role of Candida albicans homologous recombination factors Rad54 and Rdh54 in DNA damage sensitivity
Hoot, Samantha J; Zheng, Xiuzhong; Potenski, Catherine J; White, Theodore C; Klein, Hannah L
2011 ;11:214-214, BMC microbiology
ABSTRACT: BACKGROUND: The fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC), can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH) at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans. RESULTS: The data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Delta/rad54Delta mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Delta/rad54Delta mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility. CONCLUSIONS: Together, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Delta/rad54Delta mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Delta/rad54Delta and the inability to construct the double mutant rad54Delta/rad54Delta rdh54Delta/rdh54Delta suggests that Rdh54 can partially compensate for Rad54 during mitotic growth
— id: 139736, year: 2011, vol: 11, page: 214, stat: Journal Article,

R We There Yet? R-Loop Hazards to Finishing the Journey
Potenski C.J.; Klein H.L.
2011 ;44(6):848-850, Molecular cell
RNA:DNA hybrids in the genome are constantly being generated as a by-product of transcription; in this issue, two papers, from Helmrich et al. (2011) and Wahba et al. (2011), provide insight into how RNA:DNA hybrids lead to genetic instability. 2011 Elsevier Inc
— id: 148747, year: 2011, vol: 44, page: 848, stat: Journal Article,

Molecular biology: The expanding arena of DNA repair
Potenski, Catherine J; Klein, Hannah L
2011 Mar 3;471(7336):48-49, Nature
— id: 134138, year: 2011, vol: 471, page: 48, stat: Journal Article,

Methods to study mitotic homologous recombination and genome stability
Zheng, Xiuzhong; Epstein, Anastasiya; Klein, Hannah L
2011 ;745:3-13, Methods in molecular biology
Spontaneous mitotic recombination occurs in response to DNA damage incurred during DNA replication or from lesions that do not block replication but leave recombinogenic substrates such as single-stranded DNA gaps. Other types of damages result in general genome instability such as chromosome loss, chromosome fragmentation, and chromosome rearrangements. The genome is kept intact through recombination, repair, replication, checkpoints, and chromosome organization functions. Therefore when these pathways malfunction, genomic instabilities occur. Here we outline some general strategies to monitor a subset of the genomic instabilities: spontaneous mitotic recombination and chromosome loss, in both haploid and diploid cells. The assays, while not inclusive of all genome instability assays, give a broad assessment of general genome damage or inability to repair damage in various genetic backgrounds
— id: 134456, year: 2011, vol: 745, page: 3, stat: Journal Article,

Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair
Saponaro, Marco; Callahan, Devon; Zheng, Xiuzhong; Krejci, Lumir; Haber, James E; Klein, Hannah L; Liberi, Giordano
2010 Feb;6(2):e1000858-e1000858, PLoS Genetics
Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair
— id: 107900, year: 2010, vol: 6, page: e1000858, stat: Journal Article,

Swi2/Snf2-related translocases prevent accumulation of toxic Rad51 complexes during mitotic growth
Shah, Parisha P; Zheng, Xiuzhong; Epshtein, Anastasiya; Carey, Jeffrey N; Bishop, Douglas K; Klein, Hannah L
2010 Sep 24;39(6):862-872, Molecular cell
Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here, we show that Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, Rad54, and a third Swi2/Snf2 homolog Uls1 accumulated Rad51 foci, grew slowly, and suffered chromosome loss. Thus, Uls1 and Rad54 can partially substitute for Rdh54 in the removal of toxic, nondamage-associated Rad51-DNA complexes. Additional data suggest that the function of Rdh54 and Rad54 in removal of Rad51 foci is significantly specialized; Rad54 predominates for removal of damage-associated foci, and Rdh54 predominates for removal of nondamage-associated foci
— id: 147665, year: 2010, vol: 39, page: 862, stat: Journal Article,

Functional interactions of meiotic recombination factors Rdh54 and Dmc1
Chi, Peter; Kwon, Youngho; Moses, Dana N; Seong, Changhyun; Sehorn, Michael G; Singh, Akhilesh K; Tsubouchi, Hideo; Greene, Eric C; Klein, Hannah L; Sung, Patrick
2009 Feb 1;8(2):279-284, DNA repair (Amsterdam)
Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54-Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54-Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1
— id: 107905, year: 2009, vol: 8, page: 279, stat: Journal Article,

Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
Colavito, Sierra; Macris-Kiss, Margaret; Seong, Changhyun; Gleeson, Olive; Greene, Eric C; Klein, Hannah L; Krejci, Lumir; Sung, Patrick
2009 Nov;37(20):6754-6764, Nucleic acids research
The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity
— id: 107901, year: 2009, vol: 37, page: 6754, stat: Journal Article,

Breaking up just got easier to do
Klein, Hannah L; Symington, Lorraine S
2009 Jul 10;138(1):20-22, Cell
The SLX4 protein functions as a platform for catalytic subunits of structure-specific endonucleases. Findings reported in Cell (Fekairi et al., 2009; Svendsen et al., 2009) and in Molecular Cell (Andersen et al., 2009; Munoz et al., 2009) now identify the human SLX4 and show that in association with the SLX1 endonuclease it directs the symmetric cleavage and resolution of Holliday junctions
— id: 100674, year: 2009, vol: 138, page: 20, stat: Journal Article,

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination
Prakash, Rohit; Satory, Dominik; Dray, Eloise; Papusha, Almas; Scheller, Jurgen; Kramer, Wilfried; Krejci, Lumir; Klein, Hannah; Haber, James E; Sung, Patrick; Ira, Grzegorz
2009 Jan 1;23(1):67-79, Genes & development
Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks
— id: 107904, year: 2009, vol: 23, page: 67, stat: Journal Article,

Structural transitions within human Rad51 nucleoprotein filaments
Robertson, Ragan B; Moses, Dana N; Kwon, YoungHo; Chan, Pamela; Chi, Peter; Klein, Hannah; Sung, Patrick; Greene, Eric C
2009 Aug 4;106(31):12688-12693, Proceedings of the National Academy of Sciences of the United States of America
Rad51 is a core component of the eukaryotic homologous recombination machinery and is responsible for key mechanistic steps during strand invasion. Higher order oligomers of Rad51 display a remarkable degree of structural variation, forming rings, compressed filaments, and elongated filaments. It is unclear whether Rad51 can transition directly between these different oligomeric structures without disassembling first into monomers. We have used single-molecule microscopy to investigate the behavior of human Rad51 assembled on double-stranded DNA. Our results show that human Rad51 can form elongated nucleoprotein filaments on DNA, but ATP hydrolysis causes a decrease in their length without concomitant dissociation of protein. Compressed Rad51 filaments can re-elongate when presented with either ATP or the non-hydrolyzable analog AMP-PNP, and these cycles of elongation and compression are reversible. A Rad51 mutant deficient in ATP hydrolysis is locked into an extended conformation that is incapable of transitioning to a compressed filament. Similarly, wild-type Rad51 bound to DNA in the presence of AMP-PNP was trapped in the elongated state. Proteins incapable of transitioning to the compressed state were also highly resistant to dissociation from the DNA. Taken together, our results indicate that nucleotide hydrolysis by human Rad51 triggers a reversible structural transition leading to filaments with reduced helical pitch
— id: 107902, year: 2009, vol: 106, page: 12688, stat: Journal Article,

Visualizing the disassembly of S. cerevisiae Rad51 nucleoprotein filaments
Robertson, Ragan B; Moses, Dana N; Kwon, YoungHo; Chan, Pamela; Zhao, Weixing; Chi, Peter; Klein, Hannah; Sung, Patrick; Greene, Eric C
2009 May 15;388(4):703-720, Journal of molecular biology
Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level
— id: 107903, year: 2009, vol: 388, page: 703, stat: Journal Article,

Molecular biology: DNA endgames
Klein, Hannah L
2008 Oct 9;455(7214):740-741, Nature
— id: 97749, year: 2008, vol: 455, page: 740, stat: Journal Article,

The consequences of Rad51 overexpression for normal and tumor cells
Klein, Hannah L
2008 May 3;7(5):686-693, DNA repair (Amsterdam)
The Rad51 recombinase is an essential factor for homologous recombination and the repair of DNA double strand breaks, binding transiently to both single stranded and double stranded DNA during the recombination reaction. The use of a homologous recombination mechanism to repair DNA damage is controlled at several levels, including the binding of Rad51 to single stranded DNA to form the Rad51 nucleofilament, which is controlled through the action of DNA helicases that can counteract nucleofilament formation. Overexpression of Rad51 in different organisms and cell types has a wide assortment of consequences, ranging from increased homologous recombination and increased resistance to DNA damaging agents to disruption of the cell cycle and apoptotic cell death. Rad51 expression is increased in p53-negative cells, and since p53 is often mutated in tumor cells, there is a tendency for Rad51 to be overexpressed in tumor cells, leading to increased resistance to DNA damage and drugs used in chemotherapies. As cells with increased Rad51 levels are more resistant to DNA damage, there is a selection for tumor cells to have higher Rad51 levels. While increased Rad51 can provide drug resistance, it also leads to increased genomic instability and may contribute to carcinogenesis
— id: 80301, year: 2008, vol: 7, page: 686, stat: Journal Article,

ATP-dependent chromatin remodeling by the Saccharomyces cerevisiae homologous recombination factor Rdh54
Kwon, YoungHo; Seong, Changhyun; Chi, Peter; Greene, Eric C; Klein, Hannah; Sung, Patrick
2008 Apr 18;283(16):10445-10452, Journal of biological chemistry
Saccharomyces cerevisiae RDH54 is a key member of the evolutionarily conserved RAD52 epistasis group of genes needed for homologous recombination and DNA double strand break repair. The RDH54-encoded protein possesses a DNA translocase activity and functions together with the Rad51 recombinase in the D-loop reaction. By chromatin immunoprecipitation (ChIP), we show that Rdh54 is recruited, in a manner that is dependent on Rad51 and Rad52, to a site-specific DNA double strand break induced by the HO endonuclease. Because of its relatedness to Swi2/Snf2 chromatin remodelers, we have asked whether highly purified Rdh54 possesses chromatin-remodeling activity. Importantly, our results show that Rdh54 can mobilize a mononucleosome along DNA and render nucleosomal DNA accessible to a restriction enzyme, indicative of a chromatin-remodeling function. Moreover, Rdh54 co-operates with Rad51 in the utilization of naked or chromatinized DNA as template for D-loop formation. We also provide evidence for a strict dependence of the chromatin-remodeling attributes of Rdh54 on its ATPase activity and N-terminal domain. Interestingly, an N-terminal deletion mutant (rdh54Delta102) is unable to promote Rad51-mediated D-loop formation with a chromatinized template, while retaining substantial activity with naked DNA. These features of Rdh54 suggest a role of this protein factor in chromatin rearrangement during DNA recombination and repair
— id: 107906, year: 2008, vol: 283, page: 10445, stat: Journal Article,

Mechanism of eukaryotic homologous recombination
San Filippo, Joseph; Sung, Patrick; Klein, Hannah
2008 ;77:229-257, Annual review of biochemistry
Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of replication forks, for telomere maintenance, and chromosome segregation in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here
— id: 107907, year: 2008, vol: 77, page: 229, stat: Journal Article,

Reversal of fortune: Rad5 to the rescue
Klein, Hannah L
2007 Oct 26;28(2):181-183, Molecular cell
In a recent issue of Molecular Cell, Blastyak et al. (2007) show that the yeast Rad5 protein can promote error-free template switching and replication past a DNA lesion via a novel DNA unwinding reaction that also pairs nascent and parental strands
— id: 75480, year: 2007, vol: 28, page: 181, stat: Journal Article,

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling
Kwon, Youngho; Chi, Peter; Roh, Dong Hyun; Klein, Hannah; Sung, Patrick
2007 Oct 1;6(10):1496-1506, DNA repair (Amsterdam)
Rad54, a member of the Swi2/Snf2 protein family, works in concert with the RecA-like recombinase Rad51 during the early and late stages of homologous recombination. Rad51 markedly enhances the activities of Rad54, including the induction of topological changes in DNA and the remodeling of chromatin structure. Reciprocally, Rad54 promotes Rad51-mediated DNA strand invasion with either naked or chromatinized DNA. Here, using various Saccharomyces cerevisiae rad51 and rad54 mutant proteins, mechanistic aspects of Rad54/Rad51-mediated chromatin remodeling are defined. Disruption of the Rad51-Rad54 complex leads to a marked attenuation of chromatin remodeling activity. Moreover, we present evidence that assembly of the Rad51 presynaptic filament represents an obligatory step in the enhancement of the chromatin remodeling reaction. Interestingly, we find a specific interaction of the N-terminal tail of histone H3 with Rad54 and show that the H3 tail interaction domain resides within the amino terminus of Rad54. These results suggest that Rad54-mediated chromatin remodeling coincides with DNA homology search by the Rad51 presynaptic filament and that this process is facilitated by an interaction of Rad54 with histone H3
— id: 107908, year: 2007, vol: 6, page: 1496, stat: Journal Article,

Yeast recombination factor Rdh54 functionally interacts with the Rad51 recombinase and catalyzes Rad51 removal from DNA
Chi, Peter; Kwon, Youngho; Seong, Changhyun; Epshtein, Anastasiya; Lam, Isabel; Sung, Patrick; Klein, Hannah L
2006 Sep 8;281(36):26268-26279, Journal of biological chemistry
The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic inter-homologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. Here we describe an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, we demonstrate that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. We furnish evidence that the Rad51 binding domain resides within the amino-terminus of Rdh54. Accordingly, amino-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54 mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54-Rad51 complex formation. The Rdh54 expression and purification procedures described here should facilitate the functional dissection of this DNA recombination/repair factor
— id: 67273, year: 2006, vol: 281, page: 26268, stat: Journal Article,

A SUMOry of DNA replication: synthesis, damage, and repair
Klein, Hannah L
2006 Nov 3;127(3):455-457, Cell
Recombination at stalled replication forks is regulated at an early stage by sumoylation. In this issue of Cell, Branzei et al. show that the Ubc9/SUMO modification pathway controls the accumulation of cruciform structures at stalled forks
— id: 69601, year: 2006, vol: 127, page: 455, stat: Journal Article,

Mechanism of homologous recombination: mediators and helicases take on regulatory functions
Sung, Patrick; Klein, Hannah
2006 Oct;7(10):739-750, Nature reviews. Molecular cell biology
Homologous recombination (HR) is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome maintenance. As such, HR is indispensable for genome integrity, but it must be regulated to avoid deleterious events. Mutations in the tumour-suppressor protein BRCA2, which has a mediator function in HR, lead to cancer formation. DNA helicases, such as Bloom's syndrome protein (BLM), regulate HR at several levels, in attenuating unwanted HR events and in determining the outcome of HR. Defects in BLM are also associated with the cancer phenotype. The past several years have witnessed dramatic advances in our understanding of the mechanism and regulation of HR
— id: 107909, year: 2006, vol: 7, page: 739, stat: Journal Article,

Biochemical and genetic characterization of Hmi1p, a yeast DNA helicase involved in the maintenance of mitochondrial DNA
Monroe, Danny S Jr; Leitzel, Adelaide K; Klein, Hannah L; Matson, Steven W
2005 Dec;22(16):1269-1286, Yeast
The HMI1 gene encodes a DNA helicase that localizes to the mitochondria and is required for maintenance of the mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae. Identified based on its homology with E. coli uvrD, the HMI1 gene product, Hmi1p, has been presumed to be involved in the replication of the 80 kb linear S. cerevisiae mtDNA genome. Here we report the purification of Hmi1p to apparent homogeneity and provide a characterization of the helicase reaction and the ATPase reaction with regard to NTP preference, divalent cation preference and the stimulatory effects of different nucleic acids on Hmi1p-catalysed ATPase activity. Genetic complementation assays indicate that mitochondrial localization of Hmi1p is essential for its role in mtDNA metabolism. The helicase activity, however, is not essential. Point mutants that lack ATPase/helicase activity partially complement a strain lacking Hmi1p. We suggest several possible roles for Hmi1p in mtDNA metabolism
— id: 67274, year: 2005, vol: 22, page: 1269, stat: Journal Article,

Role of ATP hydrolysis in the antirecombinase function of Saccharomyces cerevisiae Srs2 protein
Krejci, Lumir; Macris, Margaret; Li, Ying; Van Komen, Stephen; Villemain, Jana; Ellenberger, Thomas; Klein, Hannah; Sung, Patrick
2004 May 28;279(22):23193-23199, Journal of biological chemistry
Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded (ss) DNA-dependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Delta or rad54Delta mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions
— id: 67276, year: 2004, vol: 279, page: 23193, stat: Journal Article,

Effects of tumor-associated mutations on Rad54 functions
Smirnova, Marina; Van Komen, Stephen; Sung, Patrick; Klein, Hannah L
2004 Jun 4;279(23):24081-24088, Journal of biological chemistry
Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous recombination and DNA double strand break repair. Rad54 belongs to the Snf2/Swi2 protein family, and it possesses a robust DNA-dependent ATPase activity, uses free energy from ATP hydrolysis to supercoil DNA, and cooperates with the Rad51 recombinase in DNA joint formation. There are two RAD54-homologous genes in human cells, hRAD54 and RAD54B. Mutations in these human genes have been found in tumors. These tumor-associated mutations map to conserved regions of the hRad54 and hRad54B proteins. Here we introduced the equivalent mutations into the Saccharomyces cerevisiae RAD54 gene in an effort to examine the functional consequences of these gene changes. One mutant, rad54 G484R, showed sensitivity to DNA-damaging agents and reduced homologous recombination rates, indicating a loss of function. Even though the purified rad54 G484R mutant protein retained the ability to bind DNA and interact with Rad51, it was nearly devoid of ATPase activity and was similarly defective in DNA supercoiling and D-loop formation. Two other mutants, rad54 N616S and rad54 D442Y, were not sensitive to genotoxic agents and behaved like the wild type allele in homologous recombination assays. Consistent with the mild phenotype associated with the rad54 N616S allele, its encoded protein was similar to wild type Rad54 protein in biochemical attributes. Because dysfunctional homologous recombination gives rise to genome instability, our results are consistent with the premise that tumor-associated mutations in hRad54 and Rad54B could contribute to the tumor phenotype or enhance the genome instability seen in tumor cells
— id: 48199, year: 2004, vol: 279, page: 24081, stat: Journal Article,

Mrc1 is required for sister chromatid cohesion to aid in recombination repair of spontaneous damage
Xu, Hong; Boone, Charles; Klein, Hannah L
2004 Aug;24(16):7082-7090, Molecular & cellular biology
The SRS2 gene of Saccharomyces cerevisiae encoding a 3'-->5' DNA helicase is part of the postreplication repair pathway and functions to ensure proper repair of DNA damage arising during DNA replication through pathways that do not involve homologous recombination. Through a synthetic gene array analysis, genes that are essential when Srs2 is absent have been identified. Among these are MRC1, TOF1, and CSM3, which mediate the intra-S checkpoint response. srs2 Delta mrc1 Delta synthetic lethality is due to inappropriate recombination, as the lethality can be suppressed by genetic elimination of homologous recombination. srs2 Delta mrc1 Delta synthetic lethality is dependent on the role of Mrc1 in DNA replication but independent of the role of Mrc1 in a DNA damage checkpoint response. mrc1 Delta, tof1 Delta and csm3 Delta mutants have sister chromatid cohesion defects, implicating sister chromatid cohesion established at the replication fork as an important factor in promoting repair of stalled replication forks through gap repair
— id: 67275, year: 2004, vol: 24, page: 7082, stat: Journal Article,

Psychiatric aspects of child and adolescent obesity: a review of the past 10 years
Zametkin, Alan J; Zoon, Christine K; Klein, Hannah W; Munson, Suzanne
2004 Feb;43(2):134-150, Journal of the American Academy of Child & Adolescent Psychiatry
OBJECTIVE: To review the past 10 years of published research on psychiatric aspects of child and adolescent obesity and highlight information mental health professionals need for preventing obesity in youths and diagnosing and treating it. METHOD: Researchers performed computerized and manual searches of the literature and summarized the most relevant articles. RESULTS: The growing epidemic of child and adolescent obesity deserves attention for its immediate mental health and long-term medical complications. Mental health professionals working with obese youths should be aware of recent advances in neuroscience, genetics, and etiologies associated with obesity. Those who assess and treat obese youth should view obesity as a chronic disease. Currently, no approved pharmacological or surgical approaches exist to treat childhood obesity. CONCLUSIONS: Health care providers should focus on modest weight-loss goals that correlate with significant health benefits. The most effective treatments include substantial parental involvement. Mental health professionals should help obese children build self-esteem to help them lead full lives regardless of weight
— id: 147666, year: 2004, vol: 43, page: 134, stat: Journal Article,

DNA helicase Srs2 disrupts the Rad51 presynaptic filament
Krejci, Lumir; Van Komen, Stephen; Li, Ying; Villemain, Jana; Reddy, Mothe Sreedhar; Klein, Hannah; Ellenberger, Thomas; Sung, Patrick
2003 May 15;423(6937):305-309, Nature
Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing
— id: 36568, year: 2003, vol: 423, page: 305, stat: Journal Article,

ATPase and DNA Helicase Activities of the Saccharomyces cerevisiae Anti-recombinase Srs2
Van Komen, Stephen; Reddy, Mothe Sreedhar; Krejci, Lumir; Klein, Hannah; Sung, Patrick
2003 Nov 7;278(45):44331-44337, Journal of biological chemistry
Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3' ss overhang, and approximately 10 bases of 3' overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3' overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the single-strand DNA-binding factor replication protein A
— id: 39080, year: 2003, vol: 278, page: 44331, stat: Journal Article,

Replication, recombination, and repair: going for the gold
Klein, Hannah L; Kreuzer, Kenneth N
2002 Mar;9(3):471-480, Molecular cell
DNA recombination is now appreciated to be integral to DNA replication and cell survival. Recombination allows replication to successfully maneuver through the roadblocks of damaged or collapsed replication forks. The signals and controls that permit cells to transition between replication and recombination modes are now being identified
— id: 27572, year: 2002, vol: 9, page: 471, stat: Journal Article,

hpr1Delta affects ribosomal DNA recombination and cell life span in Saccharomyces cerevisiae
Merker, Robert J; Klein, Hannah L
2002 Jan;22(2):421-429, Molecular & cellular biology
Multiple genetic pathways have been shown to regulate life span and aging in the yeast Saccharomyces cerevisiae. Here we show that loss of a component of the RNA polymerase II complex, Hpr1p, results in a decreased life span. Although hpr1Delta mutants have an increased rate of recombination within the ribosomal DNA (rDNA) array, this is not accompanied by an increase in extrachromosomal rDNA circles (ERCs). Analyses of mutants that affect replication of the rDNA array and suppressors that reverse the phenotypes of the hpr1Delta mutant show that the reduced life span is associated with increased genomic instability but not with increased ERC formation. The hpr1Delta mutant acts in a pathway distinct from previously described mutants that reduce life span
— id: 26519, year: 2002, vol: 22, page: 421, stat: Journal Article,

High-Copy-Number Expression of Sub2p, a Member of the RNA Helicase Superfamily, Suppresses hpr1-Mediated Genomic Instability
Fan HY; Merker RJ; Klein HL
2001 Aug;21(16):5459-5470, Molecular & cellular biology
We report on a novel role for a pre-mRNA splicing component in genome stability. The Hpr1 protein, a component of an RNA polymerase II complex and required for transcription elongation, is also required for genome stability. Deletion of HPR1 results in a 1,000-fold increase in genome instability, detected as direct-repeat instability. This instability can be suppressed by the high-copy-number SUB2 gene, which is the Saccharomyces cerevisiae homologue of the human splicing factor hUAP56. Although SUB2 is essential, conditional alleles grown at the permissive temperature complement the essential function of SUB2 yet reveal nonessential phenotypes. These studies have uncovered a role for SUB2 in preventing genome instability. The genomic instability observed in sub2 mutants can be suppressed by high-copy-number HPR1. A deletion mutant of CDC73, a component of a PolII complex, is also unstable for direct repeats. This too is suppressed by high-copy-number SUB2. Thus, defects in both the transcriptional machinery and the pre-mRNA splicing machinery can be sources of genome instability. The ability of a pre-mRNA splicing factor to suppress the hyperrecombination phenotype of a defective PolII complex raises the possibility of integrating transcription, RNA processing, and genome stability or a second role for SUB2
— id: 21106, year: 2001, vol: 21, page: 5459, stat: Journal Article,

Mutations in Recombinational Repair and in Checkpoint Control Genes Suppress the Lethal Combination of srs2Delta With Other DNA Repair Genes in Saccharomyces cerevisiae
Klein HL
2001 Feb;157(2):557-565, Genetics
The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss
— id: 17562, year: 2001, vol: 157, page: 557, stat: Journal Article,

Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast
Hegde V; Klein H
2000 Jul 15;28(14):2779-2783, Nucleic acids research
Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism
— id: 11585, year: 2000, vol: 28, page: 2779, stat: Journal Article,

A radical solution to death
Klein HL
2000 Jun;25(2):132-134, Nature genetics
— id: 17414, year: 2000, vol: 25, page: 132, stat: Journal Article,

Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1
Petukhova, G; Sung, P; Klein, H
2000 Sep 1;14(17):2206-2215, Genes & development
The first DNA joint formed in homologous recombination processes is a D-loop. Saccharomyces cerevisiae RDH54/TID1-encoded product, a Swi2/Snf2-like factor involved in recombination, is shown here to promote D-loop formation with Rad51 recombinase. Physical interaction between Rdh54 and Rad51 is functionally important because Rdh54 does not enhance the recombinase activity of the Escherichia coli RecA protein. Robust dsDNA-activated ATPase activity in Rdh54 generates unconstrained negative and positive supercoils in DNA. Efficient D-loop formation occurs with even topologically relaxed DNA, suggesting that via specific protein-protein interactions, the negative supercoils produced by Rdh54 are used by Rad51 for making DNA joints
— id: 147667, year: 2000, vol: 14, page: 2206, stat: Journal Article,

A novel cold-sensitive allele of the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase, affects the morphology of the yeast vacuole through acylation of Vac8p
Schneiter R; Guerra CE; Lampl M; Tatzer V; Zellnig G; Klein HL; Kohlwein SD
2000 May;20(9):2984-2995, Molecular & cellular biology
The yeast vacuole functions both as a degradative organelle and as a storage depot for small molecules and ions. Vacuoles are dynamic reticular structures that appear to alternately fuse and fragment as a function of growth stage and environment. Vac8p, an armadillo repeat-containing protein, has previously been shown to function both in vacuolar inheritance and in protein targeting from the cytoplasm to the vacuole. Both myristoylation and palmitoylation of Vac8p are required for its efficient localization to the vacuolar membrane (Y.-X. Wang, N. L. Catlett, and L. S. Weisman, J. Cell Biol. 140:1063-1074, 1998). We report that mutants with conditional defects in the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase (ACC1), display unusually multilobed vacuoles, similar to those observed in vac8 mutant cells. This vacuolar phenotype of acc1 mutant cells was shown biochemically to be accompanied by a reduced acylation of Vac8p which was alleviated by fatty acid supplementation. Consistent with the proposed defect of acc1 mutant cells in acylation of Vac8p, vacuolar membrane localization of Vac8p was impaired upon shifting acc1 mutant cells to nonpermissive condition. The function of Vac8p in protein targeting, on the other hand, was not affected under these conditions. These observations link fatty acid synthesis and availability to direct morphological alterations of an organellar membrane
— id: 17415, year: 2000, vol: 20, page: 2984, stat: Journal Article,

A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling
Chang M; French-Cornay D; Fan HY; Klein H; Denis CL; Jaehning JA
1999 Feb;19(2):1056-1067, Molecular & cellular biology
Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160-1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Delta ccr4Delta, paf1Delta hpr1Delta, ccr4Delta hpr1Delta, and ccr4Delta gal11Delta double mutants. In addition, paf1Delta and ccr4Delta are lethal in combination with srb5Delta, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Delta, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Delta and ccr4Delta mutations, as well as gal11Delta, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Delta mpk1Delta and paf1Delta pkc1Delta double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Delta strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes
— id: 7937, year: 1999, vol: 19, page: 1056, stat: Journal Article,

Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation
Petukhova G; Van Komen S; Vergano S; Klein H; Sung P
1999 Oct 8;274(41):29453-29462, Journal of biological chemistry
Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA
— id: 20352, year: 1999, vol: 274, page: 29453, stat: Journal Article,

The Saccharomyces cerevisiae hyperrecombination mutant hpr1Delta is synthetically lethal with two conditional alleles of the acetyl coenzyme A carboxylase gene and causes a defect in nuclear export of polyadenylated RNA
Schneiter R; Guerra CE; Lampl M; Gogg G; Kohlwein SD; Klein HL
1999 May;19(5):3415-3422, Molecular & cellular biology
In a screen for mutants that display synthetic lethal interaction with hpr1Delta, a hyperrecombination mutant of Saccharomyces cerevisiae, we have isolated a novel cold-sensitive allele of the acetyl coenzyme A (CoA) carboxylase gene, acc1(cs), encoding the rate-limiting enzyme of fatty acid synthesis. The synthetic lethal phenotype of the acc1(cs) hpr1Delta double mutant was only partially complemented by exogenous fatty acids. hpr1Delta was also synthetically lethal with a previously isolated, temperature-sensitive allele of ACC1, mtr7 (mRNA transport), indicating that the lethality of the acc1(cs) hpr1Delta double mutant was not allele specific. The basis for the interaction between conditional acc1 alleles and hpr1Delta was investigated in more detail. In the hpr1Delta mutant background, acetyl-CoA carboxylase enzyme activity was reduced about 15-fold and steady-state levels of biotinylated Acc1p and ACC1 mRNA were reduced 2-fold. The reduced Acc1p activity in hpr1Delta cells, however, did not result in an altered lipid or fatty acid composition of the mutant membranes but rendered cells hypersensitive to soraphen A, an inhibitor of Acc1p. Similar to mtr7, hpr1Delta and acc1(cs) mutant cells displayed a defect in nuclear export of polyadenylated RNA. Oversized transcripts were detected in hpr1Delta, and rRNA processing was disturbed, but pre-mRNA splicing appeared wild type. Surprisingly, the transport defect of hpr1Delta and acc1(cs) mutant cells was accompanied by an altered ring-shaped structure of the nucleolus. These observations suggest that the basis for the synthetic lethal interaction between hpr1Delta and acc1 may lie in a functional overlap of the two mutations in nuclear poly(A)+ RNA production and export that results in an altered structure of the nucleolus
— id: 8509, year: 1999, vol: 19, page: 3415, stat: Journal Article,

Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin
Vainberg IE; Lewis SA; Rommelaere H; Ampe C; Vandekerckhove J; Klein HL; Cowan NJ
1998 May 29;93(5):863-873, Cell
We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins
— id: 7835, year: 1998, vol: 93, page: 863, stat: Journal Article,

RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis
Klein HL
1997 Dec;147(4):1533-1543, Genetics
Most mitotic recombination and repair genes of Saccharomyces cerevisiae show no specificity of action for the genome ploidy. We describe here a novel repair and recombination gene that is specific for recombination and repair between homologous chromosomes. The RDH54 gene is homologous to the RAD54 gene, but rdh54 mutants do not show sensitivity to methyl methanesulfonate at concentrations that sensitize a rad54 mutant. However, the rdh54 null mutation enhances the methyl methanesulfonate sensitivity of a rad54 mutant and single rdh54 mutants are sensitive to prolonged exposure at high concentrations of methyl methanesulfonate. The RDH54 gene is required for recombination, but only in a diploid. We present evidence showing that the RDH54 gene is required for interhomologue gene conversion but not intrachromosomal gene conversion. The rdh54 mutation confers diploid-specific lethalities and reduced growth in various mutant backgrounds. These phenotypes are due to attempted recombination. The RDH54 gene is also required for meiosis as homozygous mutant diploids show very poor sporulation and reduced spore viability. The role of the RDH54 gene in mitotic repair and in meiosis and the pathway in which it acts are discussed
— id: 12190, year: 1997, vol: 147, page: 1533, stat: Journal Article,

Mutations in the RNA polymerase II transcription machinery suppress the hyperrecombination mutant hpr1 delta of Saccharomyces cerevisiae
Fan HY; Cheng KK; Klein HL
1996 Mar;142(3):749-759, Genetics
The soh1, soh2 and soh4 mutants were isolated as suppressors of the temperature-dependent growth of the hyperrecombination mutant hpr1 of Saccharomyces cerevisiae. Cloning and sequence analysis of these suppressor genes has unexpectedly shown them to code for components of the RNA polymerase II transcription complex. SOH2 is identical to RPB2, which encodes the second largest subunit of RNA polymerase II, and SOH4 is the same as SUA7, encoding the yeast transcription initiation factor TFIIB. SOH1 encodes a novel 14-kD protein with limited sequence similarity to RNA polymerases. Interestingly, SOH1 not only interacts with factors involved in DNA repair, but transcription as well. Thus, the Soh1 protein may serve to couple these two processes. The Soh1 protein interacts with a DNA repair protein, Rad5p, in a two-hybrid system assay. Soh1p may functionally interact with components of the RNA polymerase II complex as suggested from the synthetic lethality observed in soh1 rpb delta 104, soh1 soh2-1 (rpb2), and soh1 soh4 (sua7) double mutants. Because mutations in SOH1, RPB2 and SUA7 suppress the hyperrecombination phenotype of hpr1 mutants, this suggests a link between recombination in direct repeats and transcription
— id: 8417, year: 1996, vol: 142, page: 749, stat: Journal Article,

MUTATIONS IN BASAL TRANSCRIPTION FACTORS SUPPRESS THE HPR1 HYPERREC MUTANT OF YEAST
FAN, HY; KLEIN, H
1995 MAR 10 ;14(1-6):312-312, Journal of cellular biochemistry
— id: 86748, year: 1995, vol: 14, page: 312, stat: Journal Article,

Examination of mitotic recombination by means of hyper-recombination mutants in Saccharomyces cerevisiae
Klein HL
1995 ;51:271-303, Progress in nucleic acid research & molecular biology
— id: 6836, year: 1995, vol: 51, page: 271, stat: Journal Article,

Genetic control of intrachromosomal recombination
Klein HL
1995 Feb;17(2):147-159, Bioessays
Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units. Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery. Spontaneous deletions between direct repeats are generally recA-independent in E. coli and RAD52-independent in S. cerevisiae. This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process. Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process. The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae. RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate
— id: 8064, year: 1995, vol: 17, page: 147, stat: Journal Article,

RECOMBINATION IN YEAST AND ITS RELATIONSHIP TO DNA-REPAIR
KLEIN, HL; FAN, HY; CHANG, K; DIMOVA, D; THANGAVELU, A; DEMURO, MA
1995 MAR 10 ;14(1-6):269-269, Journal of cellular biochemistry
— id: 86746, year: 1995, vol: 14, page: 269, stat: Journal Article,

Characterization of mutations that suppress the temperature-sensitive growth of the hpr1 delta mutant of Saccharomyces cerevisiae
Fan HY; Klein HL
1994 Aug;137(4):945-956, Genetics
The hpr1 delta 3 mutant of Saccharomyces cerevisiae is temperature-sensitive for growth at 37 degrees and has a 1000-fold increase in deletion of tandem direct repeats. The hyperrecombination phenotype, measured by deletion of a leu2 direct repeat, is partially dependent on the RAD1 and RAD52 gene products, but mutations in these RAD genes do not suppress the temperature-sensitive growth phenotype. Extragenic suppressors of the temperature-sensitive growth have been isolated and characterized. The 14 soh (suppressor of hpr1) mutants recovered represent eight complementation groups, with both dominant and recessive soh alleles. Some of the soh mutants suppress hpr1 hyperrecombination and are distinct from the rad mutants that suppress hpr1 hyperrecombination. Comparisons between the SOH genes and the RAD genes are presented as well as the requirement of RAD genes for the Soh phenotypes. Double soh mutants have been analyzed and reveal three classes of interactions: epistatic suppression of hpr1 hyperrecombination, synergistic suppression of hpr1 hyperrecombination and synthetic lethality. The SOH1 gene has been cloned and sequenced. The null allele is 10-fold increased for recombination as measured by deletion of a leu2 direct repeat
— id: 6612, year: 1994, vol: 137, page: 945, stat: Journal Article,

Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae
Rong L; Klein HL
1993 Jan 15;268(2):1252-1259, Journal of biological chemistry
The SRS2 gene of Saccharomyces cerevisiae was identified through mutational analysis as a suppressor of radiation-sensitive mutations in the error-prone repair pathway and by a hyper-recombination phenotype. Comparison of the derived amino acid sequence revealed the gene to have high homology to the bacterial DNA helicases UvrD and Rep (Aboussekhra, A., Chanet, R., Zgaga, Z., Cassier-Chauvat, C., Heude, M., and Fabre, F. (1989) Nucleic Acids Res. 17, 7211-7219). We have purified the SRS2 protein from Escherichia coli extracts by tagging the SRS2 gene with 6 carboxyl-terminal histidine residues and overexpressing the tagged protein in a pET-3c vector. Extracts were passed over a metal-chelating affinity chromatography column followed by gel filtration to obtain an enriched protein fraction. Sephacryl gel filtration of pooled fractions containing the SRS2 protein yielded purified SRS2 protein by Coomassie Blue stain of SDS-polyacrylamide gel electrophoresis gels. The purified SRS2 protein was found to have in vitro DNA-dependent ATPase and DNA helicase activities. The polarity of the helicase activity was determined to be 3' to 5', the same polarity as that found for the UvrD and Rep proteins. The carboxyl-terminal region of the protein is shown to contain a sequence for nuclear localization. Expression of the SRS2 in yeast was examined and found to be extremely low
— id: 6501, year: 1993, vol: 268, page: 1252, stat: Journal Article,

Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants
Palladino F; Klein HL
1992 Sep;132(1):23-37, Genetics
The hyper-gene conversion srs2-101 mutation of the SRS2 DNA helicase gene of Saccharomyces cerevisiae has been reported to suppress the UV sensitivity of rad18 mutants. New alleles of SRS2 were recovered using this suppressor phenotype. The alleles have been characterized with respect to suppression of rad18 UV sensitivity, hyperrecombination, reduction of meiotic viability, and definition of the mutational change within the SRS2 gene. Variability in the degree of rad18 suppression and hyperrecombination were found. The alleles that showed the severest effects were found to be missense mutations within the consensus domains of the DNA helicase family of proteins. The effect of mutations in domains I (ATP-binding) and V (proposed DNA binding) are reported. Some alleles of SRS2 reduce spore viability to 50% of wild-type levels. This phenotype is not bypassed by spo13 mutation. Although the srs2 homozygous diploids strains undergo normal commitment to meiotic recombination, this event is delayed by several hours in the mutant strains and the strains appear to stall in the progression from meiosis I to meiosis II
— id: 13469, year: 1992, vol: 132, page: 23, stat: Journal Article,

The hyper-gene conversion hpr5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene
Rong L; Palladino F; Aguilera A; Klein HL
1991 Jan;127(1):75-85, Genetics
The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion. This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway. The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase. The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X. The hpr5-1 allele contains missense mutation in the putative ATP binding domain. A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms. We propose that the HPR5 gene functions in the RAD6 repair pathway
— id: 14195, year: 1991, vol: 127, page: 75, stat: Journal Article,

HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene
Aguilera A; Klein HL
1990 Apr;10(4):1439-1451, Molecular & cellular biology
The HPR1 gene has been cloned by complementation of the hyperrecombination phenotype of hpr1-1 strains by using a color assay system. HPR1 is a gene that is in single copy on chromosome IV of Saccharomyces cerevisiae, closely linked to ARO1, and it codes for a putative protein of 752 amino acids (molecular mass, 88 kilodaltons). Computer searches revealed homology (48.8% conserved homology; 24.8% identity) with the S. cerevisiae TOP1 gene in an alpha-helical stretch of 129 amino acids near the carboxy-terminal region of both proteins. The ethyl methanesulfonate-induced hpr1-1 mutation is a single-base change that produces a stop codon at amino acid 559 coding for a protein that lacks the carboxy-terminal TOP1 homologous region. Haploid strains carrying deletions of the HPR1 gene show a slightly reduced mitotic growth rate and extremely high rates of intrachromosomal excision recombination (frequency, 10 to 15%) but have a undetectable effect on rDNA recombination. Double-null mutants hpr1 top1 grow very poorly. We conclude that Hpr1 is a novel eucaryotic protein, mutation of which causes an increase in mitotic intrachromosomal excision recombination, and that it may be functionally related to an activity of the topoisomerase I protein
— id: 17418, year: 1990, vol: 10, page: 1439, stat: Journal Article,

Yeast alpha-factor and somatostatin enhance binding of [3H]estradiol to proteins in rat pancreas and Saccharomyces cerevisiae
Grossman, A; Klein, H; Becker, J M; Naider, F
1986 Sep;25(3):299-304, Journal of steroid biochemistry
Pancreatic tissue contains an [3H]estradiol-binding protein that requires a coligand in the steroid-binding reaction. The endogenous coligand appears to be the tetradecapeptide somatostatin. Yeast alpha-factor, a tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type, was found to be as effective as somatostatin in enhancing specific binding of [3H]estradiol to partially purified pancreatic protein. Supernatant fractions from yeast cells also contain an [3H]estradiol-binding protein. alpha-Factor can enhance specific binding of [3H]estradiol to such yeast fractions. Somatostatin, somatostatin analogues, and an analogue of alpha-factor enhanced binding of [3H]estradiol but did not inhibit cell growth or induce morphological changes in S. cerevisiae. Thus, it appears that coligand-requiring [3H]estradiol-binding activity and mating in yeast are not directly related
— id: 99130, year: 1986, vol: 25, page: 299, stat: Journal Article,

Clozapine, chlorpromazine, and placebo in newly hospitalized, acutely schizophrenic patients: a controlled, double-blind comparison
Shopsin, B; Klein, H; Aaronsom, M; Collora, M
1979 Jun;36(6):657-664, Archives of general psychiatry
Clozapine is a unique compound belonging to a relatively new group of antipsychotic agents, the dibenzazepines. To our knowledge, the present study represents the first double-blind, controlled comparison recorded in the United States. The data suggest that clozapine in the present population of newly admitted, acutely psychotic schizophrenic individuals, and in the doses employed, was more effective in overall improvement response, discharge rate, and ameliorating discrete symptoms across the different objective rating scales used than was chlorpromazine (Thorazine) hydrochloride. Placebo was ineffective. Unlike chlorpromazine, no extrapyramidal reactions occurred in those patients ingesting clozapine. Clozapine was also beneficial in reversing abnormal involuntary motor movements. It is an excellent anxiolytic and hypnotic agent. Sedation, hypotension, and hypersalivation are among the more common side effects observed
— id: 104966, year: 1979, vol: 36, page: 657, stat: Journal Article,

Clozapine: double-blind control trial in the treatment of acute schizophrenia [proceedings]
Shopsin, B; Klein, H; Aronson, M
1978 Apr;14(2):12-15, Psychopharmacology bulletin
— id: 104965, year: 1978, vol: 14, page: 12, stat: Journal Article,

Penfluridol: an open phase III study in acute newly admitted hospitalized schizophrenic patients
Shopsin, B; Klein, H; Gerbino, L; Selzer, G
1977 Dec 19;55(2):157-164, Psychopharmacology
An open study was carried out in 17 acutely ill, newly admitted, floridly psychotic schizophrenic patients to a city hospital in New York. Penfluridol was given on a daily basis up to doses of 120 mg and patients were rated objectively by means of different psychometric evaluations; vital signs were monitored daily as were side effects. The drug was found to be a rapid acting, well-tolerated, and highly effective antipsychotic agent within the population of patients explored and within the dose range used. It was particularly effective in acutely agitated floridly paranoid schizophrenics; a statistically significant impact was achieved by 7 days and usually within 72 h after initiating treatment. The drug appears unique in that (1) its effects are realized without the untoward and usually troublesome effects of nonspecific sedation attendant upon the use of many other 'neuroleptic' medications, and (2) even within the relatively high doses used it produced no hypotensive effects. It is concluded that this appears to be a unique antipsychotic agent and a potentially important addition to the treatment armamentarium of both acute and chronic schizophrenic individuals
— id: 104967, year: 1977, vol: 55, page: 157, stat: Journal Article,

High-dose penfluridol (SEMAP) in acute hospitalized schizophrenic patients
Shopsin, B; Klein, H; Selzer, G
1977 Jul;13(3):24-26, Psychopharmacology bulletin
— id: 105015, year: 1977, vol: 13, page: 24, stat: Journal Article,