Publications by authors named "Vincent A Sutera"

10 Publications

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Alternative complexes formed by the Escherichia coli clamp loader accessory protein HolC (x) with replication protein HolD (ψ) and repair protein YoaA.

DNA Repair (Amst) 2021 Feb 2;100:103006. Epub 2021 Feb 2.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, MS029, 415 South St., Waltham, MA, 02453, United States. Electronic address:

Efficient and faithful replication of DNA is essential for all organisms. However, the replication fork frequently encounters barriers that need to be overcome to ensure cell survival and genetic stability. Cells must carefully balance and regulate replication vs. repair reactions. In Escherichia coli, the replisome consists of the DNA polymerase III holoenzyme, including DNA polymerase, proofreading exonuclease, processivity clamp and clamp loader, as well as a fork helicase, DnaB and primase, DnaG. We provide evidence here that one component of the clamp loader complex, HolC (or χ) plays a dual role via its ability to form 2 mutually exclusive complexes: one with HolD (or ψ) that recruits the clamp-loader and hence the DNA polymerase holoenzyme and another with helicase-like YoaA protein, a DNA-damage inducible repair protein. By yeast 2 hybrid analysis, we show that two residues of HolC, F64 and W57, at the interface in the structure with HolD, are required for interaction with HolD and for interaction with YoaA. Mutation of these residues does not interfere with HolC's interaction with single-strand DNA binding protein, SSB. In vivo, these mutations fail to complement the poor growth and sensitivity to azidothymidine, a chain-terminating replication inhibitor. In support of the notion that these are exclusive complexes, co-expression of HolC, HolD and YoaA, followed by pulldown of YoaA, yields a complex with HolC but not HolD. YoaA fails to pulldown HolC-F64A. We hypothesize that HolC, by binding with SSB, can recruit the DNA polymerase III holoenzyme through HolD, or an alternative repair complex with YoaA helicase.
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http://dx.doi.org/10.1016/j.dnarep.2020.103006DOI Listing
February 2021

Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein.

PLoS Genet 2015 Nov 6;11(11):e1005651. Epub 2015 Nov 6.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America.

Elongating DNA polymerases frequently encounter lesions or structures that impede progress and require repair before DNA replication can be completed. Therefore, directing repair factors to a blocked fork, without interfering with normal replication, is important for proper cell function, and it is a process that is not well understood. To study this process, we have employed the chain-terminating nucleoside analog, 3' azidothymidine (AZT) and the E. coli genetic system, for which replication and repair factors have been well-defined. By using high-expression suppressor screens, we identified yoaA, encoding a putative helicase, and holC, encoding the Chi component of the replication clamp loader, as genes that promoted tolerance to AZT. YoaA is a putative Fe-S helicase in the XPD/RAD3 family for which orthologs can be found in most bacterial genomes; E. coli has a paralog to YoaA, DinG, which possesses 5' to 3' helicase activity and an Fe-S cluster essential to its activity. Mutants in yoaA are sensitive to AZT exposure; dinG mutations cause mild sensitivity to AZT and exacerbate the sensitivity of yoaA mutant strains. Suppression of AZT sensitivity by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Interactions of Chi with single-strand DNA binding protein (SSB) and with Psi were required to aid AZT tolerance, as was the proofreading 3' exonuclease, DnaQ. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to replication gaps and that unwinding of the nascent strand promotes repair and AZT excision. This recruitment prevents the toxicity of helicase activity and aids the handoff of repair with replication factors, ensuring timely repair and resumption of replication.
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http://dx.doi.org/10.1371/journal.pgen.1005651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636137PMC
November 2015

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events.

Genetics 2011 Jun 24;188(2):247-62. Epub 2011 Mar 24.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.
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http://dx.doi.org/10.1534/genetics.111.127746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122311PMC
June 2011

A role for nonessential domain II of initiator protein, DnaA, in replication control.

Genetics 2009 Sep 22;183(1):39-49. Epub 2009 Jun 22.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgE::Tn5-EZ seqADelta, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaADelta(96-120), ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaADelta(96-120) mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC.
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http://dx.doi.org/10.1534/genetics.109.104760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746162PMC
September 2009

RecA-independent recombination is efficient but limited by exonucleases.

Proc Natl Acad Sci U S A 2007 Jan 20;104(1):216-21. Epub 2006 Dec 20.

Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecA-independent recombination in Escherichia coli is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecA-dependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI(-) RecJ(-) strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that E. coli encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors.
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http://dx.doi.org/10.1073/pnas.0608293104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1765439PMC
January 2007

The role of replication initiation control in promoting survival of replication fork damage.

Mol Microbiol 2006 Apr;60(1):229-39

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.

Dam methylase mutants were recovered in a screen for mutants sensitive to UV irradiation or mild inhibition of replication elongation. Dam's role in tolerance of DNA damage is to provide binding sites for SeqA, because seqA mutants showed similar sensitivity that was genetically epistatic to dam. The sensitivity of seqA mutants to UV irradiation and to the replication inhibitors hydroxyurea (HU) and azidothymidine (AZT) was suppressed by alleles of dnaA that reduce the efficiency of replication initiation. These results suggest that for survival of replication fork damage, SeqA's repression of replication initiation is more important than its effects on nucleoid organization. Convergence of forks upon DNA damage is a likely explanation for seqA mutant sensitivity, because its poor survival of UV was suppressed by reducing secondary initiation through minimal medium growth. Surprisingly, growth in minimal medium reduced the ability of seqA+ strains to form colonies in the presence of low levels of AZT. Double dnaA seqA mutants exhibited plating efficiencies much superior to wild-type strains during chronic low-level AZT exposure in minimal medium. This suggests that mild inhibition of replication fork progression may actively restrain initiation such that seqA+ strains fail to recover initiation capacity after sustained conditions of replication arrest.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05093.xDOI Listing
April 2006

DNA repeat rearrangements mediated by DnaK-dependent replication fork repair.

Mol Cell 2006 Mar;21(5):595-604

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.

We propose that rearrangements between short tandem repeated sequences occur by errors made during a replication fork repair pathway involving a replication template switch. We provide evidence here that the DnaK chaperone of E. coli controls this template switch repair process. Mutants in dnaK are sensitive to replication fork damage and exhibit high expression of the SOS response, indicative of repair deficiency. Deletion and expansion of tandem repeats that occur by replication misalignment ("slippage") are also DnaK dependent. Because mutations in dnaX encoding the gamma and tau subunits of DNA polymerase III mimic dnaK phenotypes and are genetically epistatic, we propose that the DnaKJ chaperone remodels the replisome to facilitate repair. The fork remains largely intact because PriA or PriC restart proteins are not required. We also suggest that the poorly defined RAD6-RAD18-RAD5 mechanism of postreplication repair in eukaryotes occurs by an analogous mechanism to the DnaK template-switch pathway in prokaryotes.
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http://dx.doi.org/10.1016/j.molcel.2006.01.025DOI Listing
March 2006

RecJ exonuclease: substrates, products and interaction with SSB.

Nucleic Acids Res 2006 18;34(4):1084-91. Epub 2006 Feb 18.

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5'-3' direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5' single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5' tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading approximately 1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5' phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.
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http://dx.doi.org/10.1093/nar/gkj503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1373692PMC
February 2006

A bacterial G protein-mediated response to replication arrest.

Mol Cell 2005 Feb;17(4):549-60

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.

To define factors in E. coli promoting survival to replication fork stress, we isolated insertion mutants sensitive to replication inhibitors. One insertion caused partial loss of the universally conserved GTPase, obgE/yhbZ gene. Although obgE is essential for growth, our insertion allele supported viability until challenged with various replication inhibitors. A mutation designed to negate the GTPase activity of the protein produced similar phenotypes, but was genetically dominant. Synergistic genetic interactions with recA and recB suggested that chromosome breaks and regressed forks accumulate in obgE mutants. Mutants in obgE also exhibited asynchronous overreplication during normal growth, as revealed by flow cytometry. ObgE overexpression caused SeqA foci, normally localized to replication forks, to spread extensively within the cell. We propose that ObgE defines a pathway analogous to the replication checkpoint response of eukaryotes and acts in a complementary way to the RecA-dependent SOS response to promote bacterial cell survival to replication fork arrest.
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http://dx.doi.org/10.1016/j.molcel.2005.01.012DOI Listing
February 2005

Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways.

Genetics 2002 Mar;160(3):851-9

Rosenstiel Basic Medical Sciences Research Center and the Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

We have developed an assay for intermolecular crossing over between circular plasmids carrying variable amounts of homology. Screens of Escherichia coli mutants demonstrated that known recombination functions can only partially account for the observed recombination. Recombination rates increased three to four orders of magnitude as homology rose from 25 to 411 bp. Loss of recA blocked most recombination; however, RecA-independent crossing over predominated at 25 bp and could be detected at all homology lengths. Products of recA-independent recombination were reciprocal in nature. This suggests that RecA-independent recombination may involve a true break-and-join mechanism, but the genetic basis for this mechanism remains unknown. RecA-dependent crossing over occurred primarily by the RecF pathway but considerable recombination occurred independent of both RecF and RecBCD. In many respects, the genetic dependence of RecA-dependent crossing over resembled that reported for single-strand gap repair. Surprisingly, ruvC mutants, in both recA(+) and recA mutant backgrounds, scored as hyperrecombinational. This may occur because RuvC preferentially resolves Holliday junction intermediates, critical to both RecA-dependent and RecA-independent mechanisms, to the noncrossover configuration. Levels of crossing over were increased by defects in DnaB helicase and by oxidative damage, showing that damaged DNA or stalled replication can initiate genetic recombination.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462031PMC
March 2002