ABSTRACT
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.
Subject(s)
DNA Damage , DNA Repair , DNA Replication , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genomic Instability , Humans , Models, BiologicalABSTRACT
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 â¼80%, 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.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/metabolism , DNA Helicases/metabolism , Escherichia coli/enzymology , Microscopy, Fluorescence/methods , Models, Biological , RepliconABSTRACT
In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these translesion DNA synthesis DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the ß sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses, we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the ß clamp but not its catalytic activity, and is reinforced by the presence of the γ subunit of the ß clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.
Subject(s)
DNA Polymerase beta , DNA/metabolism , DNA Polymerase III/metabolism , DNA-Directed DNA Polymerase/metabolism , HoloenzymesABSTRACT
The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein-DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.
Subject(s)
DNA Topoisomerase IV , Escherichia coli Proteins , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , DNA Topoisomerase IV/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/metabolismABSTRACT
MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis-dependent DNA loop-extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein-protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability.
Subject(s)
Chromosomal Proteins, Non-Histone , Escherichia coli Proteins , Repressor Proteins , Adenosine Triphosphatases/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Bacterial/metabolism , DNA Topoisomerase IV/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Repressor Proteins/metabolismABSTRACT
Collisions between the replisome and RNA polymerases [RNAP(s)] are the main obstacle to DNA replication. These collisions can occur either head-on or co-directionally with respect to the direction of translocation of both complexes. Whereas head-on collisions require additional factors to be resolved, co-directional collisions are thought to be overcome by the replisome itself using the mRNA transcript as a primer. We show that mRNA takeover is utilized primarily after collisions with single RNAP complexes with short transcripts. Bypass of more complex transcription complexes requires the synthesis of a new primer downstream of the RNAP for the replisome to resume leading-strand synthesis. In both cases, bypass proceeds with displacement of the RNAP. Rep, Mfd, UvrD and RNase H can process the RNAP block and facilitate replisome bypass by promoting the formation of continuous leading strands. Bypass of co-directional RNAP(s) and/or R-loops is determined largely by the length of the obstacle that the replisome needs to traverse: R-loops are about equally as potent obstacles as RNAP arrays if they occupy the same length of the DNA template.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase , Multienzyme Complexes , Transcription, Genetic , Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli Proteins/metabolism , R-Loop Structures , RNA, Messenger/metabolism , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
DNA Replication/genetics , DNA-Binding Proteins/genetics , R-Loop Structures/genetics , Transcription, Genetic , Base Composition/genetics , Chromosome Structures/genetics , Chromosomes/genetics , DNA Helicases/genetics , DNA Repair/genetics , Escherichia coli/genetics , Genomic Instability/geneticsABSTRACT
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 vitro 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.
Subject(s)
Bacterial Proteins/chemistry , DNA Replication , DNA, Bacterial/chemistry , DNA, Single-Stranded/chemistry , Escherichia coli/chemistry , Proteolysis , Serine Endopeptidases/chemistry , Bacterial Proteins/metabolism , DNA, Bacterial/biosynthesis , DNA, Single-Stranded/biosynthesis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Rec A Recombinases/chemistry , Rec A Recombinases/metabolism , Serine Endopeptidases/metabolismABSTRACT
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.
Subject(s)
DNA Damage , DNA Replication Timing , DNA, Bacterial/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/metabolism , Multienzyme Complexes/metabolism , Bacterial Proteins/metabolism , DNA Polymerase III/metabolism , DNA Primase/metabolism , DNA-Directed DNA Polymerase/genetics , DnaB Helicases/metabolism , Escherichia coli/genetics , Kinetics , Multienzyme Complexes/genetics , Protein Binding , Templates, GeneticABSTRACT
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.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Directed DNA Polymerase/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Rec A Recombinases/chemistry , DNA, Bacterial/biosynthesis , DNA, Bacterial/chemistry , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Rec A Recombinases/metabolismABSTRACT
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 IIIß 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 Escherichia coli Topo III associates with the replication fork in vivo (likely via interactions with the single-stranded DNA-binding protein and the ß 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 ΔtopB 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.IMPORTANCE Topological entanglement between daughter chromosomes has to be reduced to exactly zero every time an E. coli cell divides. The enzymatic agents that accomplish this task are the topoisomerases. E. coli 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.
Subject(s)
Chromosomes, Bacterial/metabolism , DNA Topoisomerases, Type I/metabolism , DNA, Bacterial/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , Chromosomes, Bacterial/chemistry , DNA, Bacterial/chemistry , Nucleic Acid ConformationABSTRACT
Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The Escherichia coli 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 E. coli 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 E. coli 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 in vivo 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.
Subject(s)
DNA Breaks, Single-Stranded , DNA Polymerase III/metabolism , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Multienzyme Complexes/metabolism , DNA Polymerase III/genetics , DNA, Bacterial/metabolism , DNA-Directed DNA Polymerase/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Frameshift Mutation , Furans/chemistry , Furans/metabolism , Holoenzymes/genetics , Holoenzymes/metabolism , Multienzyme Complexes/genetics , Protein Multimerization , Pyrimidine Dimers/chemistry , Pyrimidine Dimers/metabolism , Replication OriginABSTRACT
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 mukB 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.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Chromosomes, Bacterial/chemistry , DNA Topoisomerase IV/chemistry , DNA, Bacterial/chemistry , DNA, Superhelical/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Multiprotein Complexes/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Bacterial/genetics , Chromosomes, Bacterial/metabolism , DNA Topoisomerase IV/genetics , DNA Topoisomerase IV/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , MutationABSTRACT
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 in vitro exhibit nucleoid decondensation in vivo.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , DNA, Bacterial/chemistry , DNA, Superhelical/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Protein Multimerization , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
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.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA, Bacterial/metabolism , DNA, Catenated/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Repressor Proteins/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Catenated/chemistry , DNA, Catenated/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Repressor Proteins/chemistry , Repressor Proteins/geneticsABSTRACT
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.
Subject(s)
Chromosome Segregation/genetics , Chromosomes, Bacterial/metabolism , DNA Topoisomerase IV/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Actins/genetics , Actins/metabolism , Binding Sites , Cell Cycle , Cytoskeleton/metabolism , DNA Topoisomerase IV/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Models, Biological , MutationABSTRACT
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.
Subject(s)
DNA Replication , DNA Topoisomerases, Type I/metabolism , Genome, Bacterial , RecQ Helicases/metabolism , DNA Replication Timing , DNA Topoisomerases, Type I/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Kinetics , Models, Molecular , Nucleic Acid Conformation , RecQ Helicases/genetics , S PhaseABSTRACT
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.
Subject(s)
DNA Replication , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Cell Death , DNA Helicases/metabolism , DNA-Directed DNA Polymerase/metabolism , Multienzyme Complexes/metabolism , Nucleoproteins/metabolism , Protein Binding , Transcription, GeneticABSTRACT
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' â 5' proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the ß clamp and act as a translesion polymerase but did not require its "little finger" domain, a secondary region of interaction with the ß 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.
Subject(s)
DNA Damage , DNA Repair/genetics , DNA Replication/genetics , Replication Origin/genetics , Biocatalysis , DNA Polymerase II/metabolism , DNA Polymerase beta/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Models, Genetic , Oligonucleotides/genetics , Oligonucleotides/metabolism , Protein Binding , Templates, GeneticABSTRACT
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.