Publications by authors named "Kenneth J Marians"

44 Publications

Replisome bypass of transcription complexes and R-loops.

Nucleic Acids Res 2020 10;48(18):10353-10367

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.
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http://dx.doi.org/10.1093/nar/gkaa741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544221PMC
October 2020

Two components of DNA replication-dependent LexA cleavage.

J Biol Chem 2020 07 8;295(30):10368-10379. Epub 2020 Jun 8.

Molecular Biology Program, Sloan Kettering Institute Memorial Sloan Kettering Cancer Center, New York, New York USA

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA-RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.
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http://dx.doi.org/10.1074/jbc.RA120.014224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383369PMC
July 2020

Dissecting DNA Compaction by the Bacterial Condensin MukB.

Methods Mol Biol 2019 ;2004:169-180

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Condensins in bacteria are one of the most important factors involved in the organization of long threads of DNA into compact chromosomes. The organization of DNA by condensins is vital to many DNA transactions including DNA repair and chromosome segregation. Although some of the activities of condensins are well studied, the mechanism of the overall process executed by condensins, DNA compaction, remains unclear. Here, we describe some of the methods used routinely in our laboratory to understand the mechanism of DNA compaction by Escherichia coli condensin MukB.
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http://dx.doi.org/10.1007/978-1-4939-9520-2_13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395567PMC
March 2020

Topoisomerase III Acts at the Replication Fork To Remove Precatenanes.

J Bacteriol 2019 04 13;201(7). Epub 2019 Mar 13.

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

The role of DNA topoisomerase III (Topo III) in bacterial cells has proven elusive. Whereas eukaryotic Top IIIα homologs are clearly involved with homologs of the bacterial DNA helicase RecQ in unraveling double Holliday junctions, preventing crossover exchange of genetic information at unscheduled recombination intermediates, and Top IIIβ homologs have been shown to be involved in regulation of various mRNAs involved in neuronal function, there is little evidence for similar reactions in bacteria. Instead, most data point to Topo III playing a role supplemental to that of topoisomerase IV in unlinking daughter chromosomes during DNA replication. In support of this model, we show that Topo III associates with the replication fork (likely via interactions with the single-stranded DNA-binding protein and the β clamp-loading DnaX complex of the DNA polymerase III holoenzyme), that the DnaX complex stimulates the ability of Topo III to unlink both catenated and precatenated DNA rings, and that Δ cells show delayed and disorganized nucleoid segregation compared to that of wild-type cells. These data argue that Topo III normally assists topoisomerase IV in chromosome decatenation by removing excess positive topological linkages at or near the replication fork as they are converted into precatenanes. Topological entanglement between daughter chromosomes has to be reduced to exactly zero every time an cell divides. The enzymatic agents that accomplish this task are the topoisomerases. possesses four topoisomerases. It has been thought that topoisomerase IV is primarily responsible for unlinking the daughter chromosomes during DNA replication. We show here that topoisomerase III also plays a role in this process and is specifically localized to the replisome, the multiprotein machine that duplicates the cell's genome, in order to do so.
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http://dx.doi.org/10.1128/JB.00563-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416919PMC
April 2019

The recombination mediator proteins RecFOR maintain RecA* levels for maximal DNA polymerase V Mut activity.

J Biol Chem 2019 01 27;294(3):852-860. Epub 2018 Nov 27.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

DNA template damage can potentially block DNA replication. Cells have therefore developed different strategies to repair template lesions. Activation of the bacterial lesion bypass DNA polymerase V (Pol V) requires both the cleavage of the UmuD subunit to UmuD' and the acquisition of a monomer of activated RecA recombinase, forming Pol V Mut. Both of these events are mediated by the generation of RecA* via the formation of a RecA-ssDNA filament during the SOS response. Formation of RecA* is itself modulated by competition with the ssDNA-binding protein (SSB) for binding to ssDNA. Previous observations have demonstrated that RecA filament formation on SSB-coated DNA can be favored in the presence of the recombination mediator proteins RecF, RecO, and RecR. We show here using purified proteins that in the presence of SSB and RecA, a stable RecA-ssDNA filament is not formed, although sufficient RecA* is generated to support some activation of Pol V. The presence of RecFOR increased RecA* generation and allowed Pol V to synthesize longer DNA products and to elongate from an unpaired primer terminus opposite template damage, also without the generation of a stable RecA-ssDNA filament.
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http://dx.doi.org/10.1074/jbc.RA118.005726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341379PMC
January 2019

Lesion Bypass and the Reactivation of Stalled Replication Forks.

Annu Rev Biochem 2018 06 3;87:217-238. Epub 2018 Jan 3.

Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA; email:

Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes has proven naive. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes, unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a result of cellular stress responses. These stalled forks are a major source of genome instability. The cell has developed many strategies for ensuring that these obstructions to DNA replication do not result in loss of genetic information, including DNA damage tolerance mechanisms such as lesion skipping, whereby the replisome jumps the lesion and continues downstream; template switching both behind template damage and at the stalled fork; and the error-prone pathway of translesion synthesis.
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http://dx.doi.org/10.1146/annurev-biochem-062917-011921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419508PMC
June 2018

The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops.

J Biol Chem 2017 10 25;292(41):16904-16920. Epub 2017 Aug 25.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065 and

MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation exhibit nucleoid decondensation .
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http://dx.doi.org/10.1074/jbc.M117.803312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641887PMC
October 2017

The MukB-topoisomerase IV interaction is required for proper chromosome compaction.

J Biol Chem 2017 10 25;292(41):16921-16932. Epub 2017 Aug 25.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB-topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA. This effect does not require the catalytic activity of topoisomerase IV. Cells carrying a mutant allele that encodes a protein that does not interact with topoisomerase IV exhibit severe nucleoid decompaction leading to chromosome segregation defects. These findings suggest that the MukB-topoisomerase IV complex may provide a scaffold for DNA condensation.
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http://dx.doi.org/10.1074/jbc.M117.803346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641886PMC
October 2017

Replisome-mediated translesion synthesis by a cellular replicase.

J Biol Chem 2017 08 22;292(33):13833-13842. Epub 2017 Jun 22.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The replisome transiently stalls at leading-strand template lesions and can either reinitiate replication downstream of the lesion or recruit specialized DNA polymerases that can bypass the lesion via translesion synthesis. Previous results had suggested that the replicase might play a role in lesion bypass, but this possibility has not been tested in reconstituted DNA replication systems. We report here that the DNA polymerase III holoenzyme in a stalled replisome can directly bypass a single cyclobutane pyrimidine dimer or abasic site by translesion synthesis in the absence of specialized translesion synthesis polymerases. Bypass efficiency was proportional to deoxynucleotide concentrations equivalent to those found and was dependent on the frequency of primer synthesis downstream of the lesion. Translesion synthesis came at the expense of lesion-skipping replication restart. Replication of a cyclobutane pyrimidine dimer was accurate, whereas replication of an abasic site resulted in mainly -1 frameshifts. Lesion bypass was accompanied by an increase in base substitution frequency for the base preceding the lesion. These findings suggest that DNA damage at the replication fork can be replicated directly by the replisome without the need to activate error-prone pathways.
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http://dx.doi.org/10.1074/jbc.M117.800441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566535PMC
August 2017

Independent and Stochastic Action of DNA Polymerases in the Replisome.

Cell 2017 Jun;169(7):1201-1213.e17

Department of Microbiology and Molecular Genetics and Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA. Electronic address:

It has been assumed that DNA synthesis by the leading- and lagging-strand polymerases in the replisome must be coordinated to avoid the formation of significant gaps in the nascent strands. Using real-time single-molecule analysis, we establish that leading- and lagging-strand DNA polymerases function independently within a single replisome. Although average rates of DNA synthesis on leading and lagging strands are similar, individual trajectories of both DNA polymerases display stochastically switchable rates of synthesis interspersed with distinct pauses. DNA unwinding by the replicative helicase may continue during such pauses, but a self-governing mechanism, where helicase speed is reduced by ∼80%, permits recoupling of polymerase to helicase. These features imply a more dynamic, kinetically discontinuous replication process, wherein contacts within the replisome are continually broken and reformed. We conclude that the stochastic behavior of replisome components ensures complete DNA duplication without requiring coordination of leading- and lagging-strand synthesis. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2017.05.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548433PMC
June 2017

MukB-mediated Catenation of DNA Is ATP and MukEF Independent.

J Biol Chem 2016 Nov 3;291(46):23999-24008. Epub 2016 Oct 3.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

Properly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.
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http://dx.doi.org/10.1074/jbc.M116.749994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104925PMC
November 2016

N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.

J Biomol Tech 2016 07 7;27(2):61-74. Epub 2016 Mar 7.

1 Microchemistry and Proteomics Core Laboratory, 2 Structural Biology and 3 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods.
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http://dx.doi.org/10.7171/jbt.16-2702-002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802820PMC
July 2016

Replisome-mediated translesion synthesis and leading strand template lesion skipping are competing bypass mechanisms.

J Biol Chem 2014 Nov 9;289(47):32811-23. Epub 2014 Oct 9.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3' → 5' proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its "little finger" domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions.
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http://dx.doi.org/10.1074/jbc.M114.613257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239630PMC
November 2014

Regression of replication forks stalled by leading-strand template damage: II. Regression by RecA is inhibited by SSB.

J Biol Chem 2014 Oct 19;289(41):28388-98. Epub 2014 Aug 19.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

Stalled replication forks are sites of chromosome breakage and the formation of toxic recombination intermediates that undermine genomic stability. Thus, replication fork repair and reactivation are essential processes. Among the many models of replication fork reactivation is one that invokes fork regression catalyzed by the strand exchange protein RecA as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecA using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that RecA is unable to regress the stalled fork in the presence of the replisome and SSB. If the replication proteins are removed from the stalled fork, RecA will catalyze net regression as long as the Okazaki fragments are sealed. RecA-generated Holliday junctions can be detected by RuvC cleavage, although this is not a robust reaction. On the other hand, extensive branch migration by RecA, where a completely unwound product consisting of the paired nascent leading and lagging strands is produced, is observed under conditions where RuvC activity is suppressed. This branch migration reaction is inhibited by SSB, possibly accounting for the failure of RecA to generate products in the presence of the replication proteins. Interestingly, we find that the RecA-RuvC reaction is supported to differing extents, depending on the template damage; templates carrying a cyclopyrimidine dimer elicit more RecA-RuvC product than those carrying a synthetic abasic site. This difference could be ascribed to a higher affinity of RecA binding to DNAs carrying a thymidine dimer than to those with an abasic site.
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http://dx.doi.org/10.1074/jbc.M114.587907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192491PMC
October 2014

Regression of replication forks stalled by leading-strand template damage: I. Both RecG and RuvAB catalyze regression, but RuvC cleaves the holliday junctions formed by RecG preferentially.

J Biol Chem 2014 Oct 19;289(41):28376-87. Epub 2014 Aug 19.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

The orderly progression of replication forks formed at the origin of replication in Escherichia coli is challenged by encounters with template damage, slow moving RNA polymerases, and frozen DNA-protein complexes that stall the fork. These stalled forks are foci for genomic instability and must be reactivated. Many models of replication fork reactivation invoke nascent strand regression as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecG and RuvAB, two proteins commonly thought to be involved in the process, using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that both RecG and RuvAB can regress the stalled fork in the presence of the replisome and SSB; however, RuvAB generates a completely unwound product consisting of the paired nascent leading and lagging strands, whereas RuvC cleaves the Holliday junction generated by RecG-catalyzed fork regression. We also find that RecG stimulates RuvAB-catalyzed regression, presumably because it is more efficient at generating the initial Holliday junction from the stalled fork.
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http://dx.doi.org/10.1074/jbc.M114.587881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192490PMC
October 2014

Dynamics of leading-strand lesion skipping by the replisome.

Mol Cell 2013 Dec 21;52(6):855-65. Epub 2013 Nov 21.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Electronic address:

The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate-limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis.
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http://dx.doi.org/10.1016/j.molcel.2013.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877186PMC
December 2013

Characterization of the nucleoid-associated protein YejK.

J Biol Chem 2013 Nov 16;288(44):31503-16. Epub 2013 Sep 16.

From the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.

Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12-14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging.
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http://dx.doi.org/10.1074/jbc.M113.494237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814747PMC
November 2013

Rescuing stalled or damaged replication forks.

Cold Spring Harb Perspect Biol 2013 May 1;5(5):a012815. Epub 2013 May 1.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

In recent years, an increasing number of studies have shown that prokaryotes and eukaryotes are armed with sophisticated mechanisms to restart stalled or collapsed replication forks. Although these processes are better understood in bacteria, major breakthroughs have also been made to explain how fork restart mechanisms operate in eukaryotic cells. In particular, repriming on the leading strand and fork regression are now established as critical for the maintenance and recovery of stalled forks in both systems. Despite the lack of conservation between the factors involved, these mechanisms are strikingly similar in eukaryotes and prokaryotes. However, they differ in that fork restart occurs in the context of chromatin in eukaryotes and is controlled by multiple regulatory pathways.
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http://dx.doi.org/10.1101/cshperspect.a012815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632063PMC
May 2013

Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli.

Proc Natl Acad Sci U S A 2013 Apr 15;110(18):7252-7. Epub 2013 Apr 15.

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom.

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.
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http://dx.doi.org/10.1073/pnas.1303890110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645559PMC
April 2013

The MukB-ParC interaction affects the intramolecular, not intermolecular, activities of topoisomerase IV.

J Biol Chem 2013 Mar 24;288(11):7653-7661. Epub 2013 Jan 24.

Molecular Biology Program, Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York 10065; Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York 10065. Electronic address:

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.
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http://dx.doi.org/10.1074/jbc.M112.418087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597806PMC
March 2013

Purification and characterization of Escherichia coli MreB protein.

J Biol Chem 2013 Feb 12;288(5):3469-75. Epub 2012 Dec 12.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.
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http://dx.doi.org/10.1074/jbc.M112.413708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561565PMC
February 2013

A role for topoisomerase III in Escherichia coli chromosome segregation.

Mol Microbiol 2012 Nov 16;86(4):1007-22. Epub 2012 Oct 16.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

The cellular function of Escherichia coli topoisomerase III remains elusive. We show that rescue of temperature-sensitive mutants in parE and parC (encoding the subunits of the chromosomal decatenase topoisomerase IV) at restrictive temperatures by high-copy suppressors is strictly dependent on topB (encoding topoisomerase III). Double mutants of parEΔtopB and parCΔtopB were barely viable, grew slowly, and were defective in chromosome segregation at permissive temperatures. The topB mutant phenotype did not result from accumulation of toxic recombination intermediates, because it was not relieved by mutations in either recQ or recA. In addition, in an otherwise wild-type genetic background, ΔtopB cells treated with the type II topoisomerase inhibitor novobiocin displayed aberrant chromosome segregation. This novobiocin sensitivity was attributable to an increased demand for topoisomerase IV and is unlikely to define a new role for topoisomerase III; therefore, these results suggest that topoisomerase III participates in orderly and efficient chromosome segregation in E. coli.
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http://dx.doi.org/10.1111/mmi.12039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549057PMC
November 2012

The Escherichia coli replisome is inherently DNA damage tolerant.

Science 2011 Oct;334(6053):235-8

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

The Escherichia coli DNA replication machinery must frequently overcome template lesions under normal growth conditions. Yet, the outcome of a collision between the replisome and a leading-strand template lesion remains poorly understood. Here, we demonstrate that a single, site-specific, cyclobutane pyrimidine dimer leading-strand template lesion provides only a transient block to fork progression in vitro. The replisome remains stably associated with the fork after collision with the lesion. Leading-strand synthesis is then reinitiated downstream of the damage in a reaction that is dependent on the primase, DnaG, but independent of any of the known replication-restart proteins. These observations reveal that the replisome can tolerate leading-strand template lesions without dissociating by synthesizing the leading strand discontinuously.
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http://dx.doi.org/10.1126/science.1209111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593629PMC
October 2011

Structure of the SSB-DNA polymerase III interface and its role in DNA replication.

EMBO J 2011 Aug 19;30(20):4236-47. Epub 2011 Aug 19.

Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA.

Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.
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http://dx.doi.org/10.1038/emboj.2011.305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199393PMC
August 2011

Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in Escherichia coli.

Proc Natl Acad Sci U S A 2010 Nov 9;107(44):18826-31. Epub 2010 Aug 9.

Weill Cornell Graduate School of Medical Sciences, Memorial Sloan–Kettering Cancer Center, New York, NY 10065, USA.

Proper geometric and topological organization of DNA is essential for all chromosomal processes. Two classes of proteins play major roles in organizing chromosomes: condensin complexes and type II topoisomerases. In Escherichia coli, MukB, a structural maintenance of chromosome-like component of the bacterial condensin, and topoisomerase IV (Topo IV), a type II topoisomerase that decatenates the newly replicated daughter chromosomes, are both essential for chromosome segregation in rapidly growing cells. However, little is known about the interplay between MukB and Topo IV. Here we demonstrate a physical and functional interaction between MukB and ParC, a subunit of Topo IV, in vitro. The site of MukB interaction was located on the C-terminal domain of ParC and a loss-of-interaction mutant, ParC R705E R729A, was isolated. This variant retained full activity as a topoisomerase when reconstituted with ParE to form Topo IV. We show that MukB stimulates the superhelical DNA relaxation activity of wild-type Topo IV, but not that of Topo IV reconstituted with ParC R705E R729A.
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http://dx.doi.org/10.1073/pnas.1008140107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2973858PMC
November 2010

Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival.

DNA Repair (Amst) 2010 Mar 22;9(3):202-9. Epub 2010 Jan 22.

Molecular Biology Program, Weill-Cornell Graduate School of Medical Sciences, New York, NY, USA.

PriA, a 3'-->5' superfamily 2 DNA helicase, acts to remodel stalled replication forks and as a specificity factor for origin-independent assembly of a new replisome at the stalled fork. The ability of PriA to initiate replication at stalled forked structures ensures complete genome replication and helps to protect the cell from illegitimate recombination events. This review focuses on the activities of PriA and its role in replication fork assembly and maintaining genomic integrity.
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http://dx.doi.org/10.1016/j.dnarep.2009.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2827650PMC
March 2010

DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

Nucleic Acids Res 2010 May 16;38(9):3031-40. Epub 2010 Jan 16.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(-)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.
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http://dx.doi.org/10.1093/nar/gkp1243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875013PMC
May 2010

Actin homolog MreB affects chromosome segregation by regulating topoisomerase IV in Escherichia coli.

Mol Cell 2009 Jan;33(2):171-80

Program in Molecular Biology, Weill Graduate School of Cornell University, New York, NY 10065, USA.

In Escherichia coli, topoisomerase IV, a type II topoisomerase, mediates the resolution of topological linkages between replicated daughter chromosomes and is essential for chromosome segregation. Topo IV activity is restricted to only a short interval late in the cell cycle. However, the mechanism that confers this temporal regulation is unknown. Here we report that the bacterial actin homolog MreB participates in the temporal oscillation of Topo IV activity. We show that mreB mutant strains are deficient in Topo IV activity. In addition, we demonstrate that, depending upon whether it is in a monomeric or polymerized state, MreB affects Topo IV activity differentially. In addition, MreB physically interacts with the ParC subunit of Topo IV. Together, these results may explain how dynamics of the bacterial cytoskeleton are coordinated with the timing of chromosome segregation.
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http://dx.doi.org/10.1016/j.molcel.2009.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759193PMC
January 2009

Resolution of converging replication forks by RecQ and topoisomerase III.

Mol Cell 2008 Jun;30(6):779-89

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.

RecQ-like DNA helicases pair with cognate topoisomerase III enzymes to function in the maintenance of genomic integrity in many organisms. These proteins play roles in stabilizing stalled replication forks, the S phase checkpoint response, and suppressing genetic crossovers, and their inactivation results in hyper-recombination, gross chromosomal rearrangements, chromosome segregation defects, and human disease. Biochemical activities associated with these enzymes include the ability to resolve double Holliday junctions, a process thought to lead to the suppression of crossover formation. Using Escherichia coli RecQ and topoisomerase III, we demonstrate a second activity for this pair of enzymes that could account for their role in maintaining genomic stability: resolution of converging replication forks. This resolution reaction is specific for the RecQ-topoisomerase III pair and is mediated by interaction of both of these enzymes with the single-stranded DNA-binding protein SSB.
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http://dx.doi.org/10.1016/j.molcel.2008.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2459239PMC
June 2008

Understanding how the replisome works.

Nat Struct Mol Biol 2008 Feb;15(2):125-7

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http://dx.doi.org/10.1038/nsmb0208-125DOI Listing
February 2008
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