James A Borowiec, Ph.D.

Specific domains of nucleolin interact with Hdm2 and antagonize Hdm2-mediated p53 ubiquitination
Bhatt, Purvi; d'Avout, Claire; Kane, Naomi S; Borowiec, James A; Saxena, Anjana
2012 Feb;279(3):370-383, FEBS journal
Nucleolin is an abundant multifunctional nucleolar protein with defined roles in ribosomal RNA processing, RNA polymerase I catalyzed transcription and the regulation of apoptosis. Earlier we reported that human nucleolin binds to the p53 antagonist human double minute 2 (Hdm2) as determined by reciprocal co-immunoprecipitation assays using cell lysates. We also demonstrated that nucleolin antagonizes Hdm2-mediated degradation of p53. Here, we identify specific domains of nucleolin and Hdm2 proteins that support mutual interaction and investigate the implications of complex formation on p53 ubiquitination and protein levels. Our data indicate that the nucleolin N-terminus as well as the central RNA-binding domain (RBD) are predominantly involved in binding to Hdm2. The nucleolin RBD robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2 in vitro, while the N-terminus of nucleolin preferentially associated with the Hdm2 RING (really interesting new gene) domain expressed in cells. We further demonstrate that the C-terminal glycine-arginine rich domain of nucleolin serves as the predominant binding domain for direct interaction with p53. While overexpression of nucleolin or its various domains had no significant effect on Hdm2 auto-ubiquitination, the nucleolin RBD antagonized the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. Conversely, the adjacent glycine-arginine rich domain of nucleolin interacted with p53 causing a modest stimulatory effect on p53 ubiquitination. These data suggest that changes in nucleolin conformation can alter the availabilities of such domains in vivo to modulate the overall impact of nucleolin on Hdm2 activity and hence on p53 stability
— id: 149953, year: 2012, vol: 279, page: 370, stat: Journal Article,

Open Sesame: activating dormant replication origins in the mouse immunoglobulin heavy chain (Igh) locus
Borowiec, James A; Schildkraut, Carl L
2011 Jun;23(3):284-292, Current opinion in cell biology
Chromosomal DNA replication in mammals initiates from replication origins whose activity differs in accordance with cell type and differentiation state. In addition to origins that are active in unperturbed conditions, chromosomes also contain dormant origins that can become functional in response to certain genotoxic stress conditions. Improper regulation of origin usage can cause genomic instability leading to tumorigenesis. We review findings from recent single-molecule DNA fiber studies examining replication of the mouse immunoglobulin heavy chain (Igh) locus, in which origin activity over a 400kb region is subject to dramatic developmental regulation. Possible models are discussed to explain such differential origin usage, particularly during replication stress conditions that can activate dormant origins
— id: 134310, year: 2011, vol: 23, page: 284, stat: Journal Article,

Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress
Brocardo M.G.; Borowiec J.A.; Henderson B.R.
2011 ;43(9):1354-1364, International journal of biochemistry & cell biology
The adenomatous polyposis coli (APC) tumor suppressor traffics between nucleus and cytoplasm to perform distinct functions. Here we identify a specific role for APC in the DNA replication stress response. The silencing of APC caused an accumulation of asynchronous cells in early S phase and delayed S phase progression in cells released from hydroxyurea-mediated replication arrest. Immunoprecipitation assays revealed a selective binding of APC to replication protein A 32 kDa subunit (RPA32), and the APC-RPA32 complex increased at chromatin after hydroxyurea treatment. Interestingly, APC knock-down prevented accumulation at chromatin of the stress-induced S33- and S29-phosphorylated forms of RPA32, and reduced the expression of ATR-phosphorylated forms of S317-phospho-Chk1 and -H2AX. Using RPA32-inducible cells we showed that reconstitution of RPA32 diminished the S-phase delay caused by loss of APC. In contrast to full-length APC, the truncated APC mutant protein expressed in SW480 colon cancer cells was impaired in its binding and regulation of RPA32, and failed to regulate cell cycle after replication stress. We propose that APC associates with RPA at stalled DNA replication forks and promotes the ATR-dependent phosphorylation of RPA32, Chk1 and -H2AX in response to DNA replication stress, thereby influencing the rate of re-entry into the cell cycle. 2011 Elsevier Ltd. All rights reserved
— id: 136528, year: 2011, vol: 43, page: 1354, stat: Journal Article,

A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination
Lee, DH; Pan, YF; Kanner, S; Sung, P; Borowiec, JA; Chowdhury, D
2010 MAR ;17(3):365-U138, Nature structural & molecular biology
Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage-dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP4R2 in cells alters the kinetics and pattern of RPA2 phosphorylation. Depletion of PP4R2 impedes homologous recombination (HR) via inefficient loading of the essential HR factor RAD51, causing an extended G2-M checkpoint and hypersensitivity to DNA damage. Cells expressing phosphomimetic RPA2 mutants have a comparable phenotype, suggesting that PP4-mediated dephosphorylation of RPA2 is necessary for an efficient DNA-damage response. These observations provide new insight into the role and regulation of RPA phosphorylation in HR-mediated repair
— id: 108314, year: 2010, vol: 17, page: 365, stat: Journal Article,

Mitotic crisis: the unmasking of a novel role for RPA
Anantha, Rachel William; Borowiec, James A
2009 Feb 1;8(3):357-361, Cell cycle
Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (2008; PNAS 105:12903-8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G(1) phase. Along with providing additional discussion of this study, we review evidence suggesting that DNA replication and repair factors can modulate mitotic transit by acting through Polo-like kinase-1 (Plk1) and the centrosome. 'A crisis unmasks everyone.'-Mason Cooley, U.S. aphorist
— id: 95184, year: 2009, vol: 8, page: 357, stat: Journal Article,

Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress
Vassin, Vitaly M; Anantha, Rachel William; Sokolova, Elena; Kanner, Shlomo; Borowiec, James A
2009 Nov 15;122(Pt 22):4070-4080, Journal of cell science
ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replication forks, with S33-P-RPA2 contained within nuclear repair centers. Although cells in which endogenous RPA2 was 'replaced' with an RPA2 protein with mutations T21A and S33A (T21A/S33A-RPA) had normal levels of DNA replication under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress
— id: 104802, year: 2009, vol: 122, page: 4070, stat: Journal Article,

RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage
Anantha, Rachel William; Sokolova, Elena; Borowiec, James A
2008 Sep 2;105(35):12903-12908, Proceedings of the National Academy of Sciences of the United States of America
Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G(1) phase after exposure to bleomycin. An increased percentage of these mutant cells also was positive initially for cyclin B expression and BubR1 chromatin staining, indicative of an extended spindle assembly checkpoint. The mutant cells that experienced mitotic DNA damage also underwent apoptosis at higher levels than cells expressing the WT subunit. Even so, we did not find the mutation had any dramatic effects on the level of DNA repair in mitosis. Cells lacking ATM (a checkpoint factor and RPA2 kinase) also were severely defective in mitotic exit and were unable to support RPA hyperphosphorylation after mitotic DNA damage. Although checkpoint 1 effector kinase (Chk1) had a more complex role, inhibition of Chk1 activity with UCN-01 also reduced mitotic exit. Chk1 activation and mitotic RPA hyperphosphorylation were found to be independent events. Our results demonstrate that mitotic RPA hyperphosphorylation facilitates release of cells from a damaged mitosis into a 2N G(1) phase, thereby increasing cell viability
— id: 87807, year: 2008, vol: 105, page: 12903, stat: Journal Article,

Sequential and synergistic modification of human Replication Factor A stimulates chromosomal DNA repair
Anantha, Rachel W; Vassin, Vitaly M; Borowiec, James A
2007 Dec 7;282(49):35910-35923, Journal of biological chemistry
The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-S29, a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in non-stressed cells. Robust phosphorylation of S29 was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. That is, the initial modification of S33 by ATR stimulates subsequent phosphorylation of Cdk sites S23 and S29. These events then facilitate modification of T21 and extreme N-terminal sites S4 and S8, likely by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was 'replaced' with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci, and had persistent staining of H2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized
— id: 74674, year: 2007, vol: 282, page: 35910, stat: Journal Article,

Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization
Saxena, A; Rorie, C J; Dimitrova, D; Daniely, Y; Borowiec, J A
2006 Nov 23;25(55):7274-7288, Oncogene
Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21(cip1/waf1), a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition
— id: 69575, year: 2006, vol: 25, page: 7274, stat: Journal Article,

Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation
Kim, Kyung; Dimitrova, Diana D; Carta, Kristine M; Saxena, Anjana; Daras, Mariza; Borowiec, James A
2005 Mar;25(6):2463-2474, Molecular & cellular biology
Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor
— id: 48733, year: 2005, vol: 25, page: 2463, stat: Journal Article,

The Toposome: A New Twist on Topoisomerase IIalpha
Borowiec, James A
2004 May;3(5):627-628, Cell cycle
— id: 42686, year: 2004, vol: 3, page: 627, stat: Journal Article,

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers
Vassin, Vitaly M; Wold, Marc S; Borowiec, James A
2004 Mar;24(5):1930-1943, Molecular & cellular biology
Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage
— id: 42117, year: 2004, vol: 24, page: 1930, stat: Journal Article,

Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation
Daniely, Yaron; Dimitrova, Diana D; Borowiec, James A
2002 Aug;22(16):6014-6022, Molecular & cellular biology
We recently discovered that heat shock causes nucleolin to relocalize from the nucleolus to the nucleoplasm, whereupon it binds replication protein A and inhibits DNA replication initiation. We report that nucleolin mobilization also occurs following exposure to ionizing radiation (IR) and treatment with camptothecin. Mobilization was selective in that another nucleolar marker, upstream binding factor, did not relocalize in response to IR. Nucleolin relocalization was dependent on p53 and stress, the latter initially stimulating nucleolin-p53 complex formation. Nucleolin relocalization and complex formation in vivo were independent of p53 transactivation but required the p53 C-terminal regulatory domain. Nucleolin and p53 also interact directly in vitro, with a similar requirement for p53 domains. These data indicate a novel p53-dependent mechanism in which cell stress mobilizes nucleolin for transient replication inhibition and DNA repair
— id: 32273, year: 2002, vol: 22, page: 6014, stat: Journal Article,

Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication
Daniely Y; Borowiec JA
2000 May 15;149(4):799-810, Journal of cell biology
We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin-hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin-hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress
— id: 11696, year: 2000, vol: 149, page: 799, stat: Journal Article,

5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates
Iftode C; Borowiec JA
2000 Oct 3;39(39):11970-11981, Biochemistry
Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) 'pseudo-origin' (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cis-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin
— id: 17118, year: 2000, vol: 39, page: 11970, stat: Journal Article,

Replication protein A (RPA): the eukaryotic SSB
Iftode C; Daniely Y; Borowiec JA
1999 ;34(3):141-180, Critical reviews in biochemistry & molecular biology
Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is highly conserved in eukaryotes. RPA plays essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair, and homologous recombination. In this review, we provide a comprehensive overview of RPA structure and function and highlight the more recent developments in these areas. The last few years have seen major advances in our understanding of the mechanism of RPA binding to DNA, including the structural characterization of the primary DNA-binding domains (DBD) and the identification of two secondary DBDs. Moreover, evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation. In addition to its mechanistic roles, RPA interacts with many key factors in nucleic acid metabolism, and we discuss the critical nature of many of these interactions to DNA metabolism. RPA is a phosphorylation target for DNA-dependent protein kinase (DNA-PK) and likely the ataxia telangiectasia-mutated gene (ATM) protein kinase, and recent observations are described that suggest that RPA phosphorylation plays a significant modulatory role in the cellular response to DNA damage
— id: 6191, year: 1999, vol: 34, page: 141, stat: Journal Article,

Distinct roles of two binding sites for the bovine papillomavirus (BPV) E2 transactivator on BPV DNA replication
Gillette TG; Borowiec JA
1998 Jul;72(7):5735-5744, Journal of virology
The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex
— id: 8266, year: 1998, vol: 72, page: 5735, stat: Journal Article,

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process
Iftode C; Borowiec JA
1998 Dec 15;26(24):5636-5643, Nucleic acids research
The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a 'pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 x 10(-7) M, compared to 9.0 x 10(-8) M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be approximately 4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37 degreesC stimulated denaturation nearly 4-fold. At 37 degreesC, denaturation occurred on approximately 1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate
— id: 7355, year: 1998, vol: 26, page: 5636, stat: Journal Article,

Synthetic DNA replication bubbles bound and unwound with twofold symmetry by a simian virus 40 T-antigen double hexamer
Smelkova NV; Borowiec JA
1998 Nov;72(11):8676-8681, Journal of virology
Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires >/=40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5' junction on each strand, with 5 bp of duplex DNA and approximately 17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5' direction into the duplex region, while resulting in no significant changes to the 3' edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex
— id: 7449, year: 1998, vol: 72, page: 8676, stat: Journal Article,