Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.     

Replication protein A is sequentially phosphorylated during meiosis

Phosphorylation of the cellular single-stranded DNA-binding protein, replication protein A (RPA), occurs during normal mitotic cell cycle progression and also in response to genotoxic stress. In budding yeast, these reactions require the ATM homolog Mec1, a central regulator of the DNA replication and DNA damage checkpoint responses. We now demonstrate that the middle subunit of yeast RPA (Rfa2) becomes phosphorylated in two discrete steps during meiosis. Primary Rfa2 phosphorylation occurs early in meiotic progression and is independent of DNA replication, recombination and Mec1. In contrast, secondary Rfa2 phosphorylation is activated upon initiation of recombination and requires Mec1. While the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to disappear soon after recombination is complete. Extensive secondary Rfa2 phosphorylation is observed in a recombination mutant defective for the pachytene checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay in response to DNA double-strand breaks. Our results suggest that Mec1-dependent RPA phosphorylation could be involved in regulating recombination rather than cell cycle or meiotic progression.     

Therefore,what are recombination proteins there for?

The order of discovery can have a profound effect upon the way in which we think about the function of a gene. In E. coli, recA is nearly essential for cell survival in the presence of DNA damage. However, recA was originally identified, as a gene required to obtain recombinant DNA molecules in conjugating bacteria. As a result, it has been frequently assumed that recA promotes the survival of bacteria containing DNA damage by recombination in which DNA strand exchanges occur. We now know that several of the processes that interact with or are controlled by recA, such as excision repair and translesion synthesis, operate to ensure that DNA replication occurs processively without strand exchanges. Yet the view persists in the literature that recA functions primarily to promote recombination during DNA repair. With the benefit of hindsight and more than three decades of additional research, we reexamine some of the classical experiments that established the concept of DNA repair by recombination, and we consider the possibilities that recombination is not an efficient mechanism for rescuing damaged cells, and that recA may be important for maintaining processive replication in a manner that does not generally promote recombination.     

Recombination and Replication

The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.The interplay between replication and recombination is complex in terms of both mechanism and integration within DNA metabolism. At the heart of this interplay is the requirement for single-stranded DNA (ssDNA), the substrate for DNA-strand-exchange proteins, to initiate recombination (Cox 2007b; San Filippo et al. 2008). Whether, when, and where this ssDNA is generated determines the functional relationship between replication and recombination, a relationship that can operate in both directions. Homologous recombination enzymes are critical for successful completion of genome duplication (Kogoma 1997; Cox et al. 2000) but DNA replication also underpins homologous recombination, as discussed elsewhere in this collection. The links between recombination and replication are therefore intimate and one cannot be considered in isolation from the other. However, involvement of DNA-strand-exchange proteins, regardless of the metabolic context, comes with the unavoidable risk of genome rearrangements. This genome instability can occasionally increase evolutionary fitness but more frequently is deleterious to the viability of the individual.This review will focus on fundamental aspects of the links between replication and recombination enzymes rather than simply providing a list of known enzymes and reactions. The substrate, DNA, is identical in all of these reactions and this is reflected in the high mechanistic conservation of replication and recombination.     

Replication fork barriers: pausing for a break or stalling for time?

Defects in chromosome replication can lead to translocations that are thought to result from recombination events at stalled DNA replication forks. The progression of forks is controlled by an essential DNA helicase, which unwinds the parental duplex and can stall on encountering tight protein-DNA complexes. Such pause sites are hotspots for recombination and it has been proposed that stalled replisomes disassemble, leading to fork collapse. However, in both prokaryotes and eukaryotes it now seems that paused forks are surprisingly stable, so that DNA synthesis can resume without recombination if the barrier protein is removed. Recombination at stalled forks might require other events that occur after pausing, or might be dependent on features of the surrounding DNA sequence. These findings have important implications for our understanding of the regulation of genome stability in eukaryotic cells, in which pausing of forks is mediated by specific proteins that are associated with the replicative helicase.     

Interplay of DNA repair,homologous recombination,and DNA polymerases in resistance to the DNA damaging agent 4-nitroquinoline-1-oxide in Escherichia coli

Escherichia coli has three DNA damage-inducible DNA polymerases: DNA polymerase II (Pol II), DNA polymerase IV (Pol IV), and DNA polymerase V (Pol V). While the in vivo function of Pol V is well understood, the precise roles of Pol IV and Pol II in DNA replication and repair are not as clear. Study of these polymerases has largely focused on their participation in the recovery of failed replication forks, translesion DNA synthesis, and origin-independent DNA replication. However, their roles in other repair and recombination pathways in E. coli have not been extensively examined. This study investigated how E. coli's inducible DNA polymerases and various DNA repair and recombination pathways function together to convey resistance to 4-nitroquinoline-1-oxide (NQO), a DNA damaging agent that produces replication blocking DNA base adducts. The data suggest that full resistance to this compound depends upon an intricate interplay among the activities of the inducible DNA polymerases and recombination. The data also suggest new relationships between the different pathways that process recombination intermediates.     

DNA helicase mutants of bacteriophage T4 that are defective in DNA recombination

We previously isolated a plasmid-borne, recombination-deficient mutant derivative of the bacteriophage T4 DNA helicase gene 41. We have now transferred this 41rrh1 mutation into the phage genome in order to characterize its mutational effects further. The mutation impairs a recombination pathway that is distinct from the pathway involving uvsX, which is essential for strand transfer, and it also eliminates most homologous recombination between a plasmid and the T4 genome. Although 41rrh1 does not affect T4 DNA replication from some origins, it does inactivate plasmid replication that is dependent on ori(uvsY) and ori(34), as well as recombination-dependent DNA replication. Combination of 41rrh1 with some uvsX alleles is lethal. Based on these results, we propose that gene 41 contributes to DNA recombination through its role in DNA replication. Received: 3 February 1999 / Accepted: 20 July 1999     

Recombination-dependent DNA replication in phage T4

Studies in the 1960s implied that bacteriophage T4 tightly couples DNA replication to genetic recombination. This contradicted the prevailing wisdom of the time, which staunchly supported recombination as a simple cut-and-paste process. More-recent investigations have shown how recombination triggers DNA synthesis and why the coupling of these two processes is important. Results from T4 were instrumental in our understanding of many important replication and recombination proteins, including the newly recognized replication/recombination mediator proteins. Recombination-dependent DNA replication is crucial to the T4 life cycle as it is the major mode of DNA replication and is also central to the repair of DNA breaks and other damage.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.     

Replication and recombination in adenovirus-infected cells are temporally and functionally related.

We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9 degrees C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9 degrees C.     

Links between replication, recombination and genome instability in eukaryotes

Double-strand breaks in DNA can be repaired by homologous recombination including break-induced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.     

Initiation of genetic recombination and recombination-dependent replication

Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.     

Recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.     

Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.     

Proliferating cell nuclear antigen: More than a clamp for DNA polymerases

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases δ and ε. It now appears that proliferating cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.     

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Delta double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Delta alone. However, surprisingly, the dna2-2 sgs1Delta lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Delta lethality is only partially suppressed by deletion of rad51Delta. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.     

A dynamic RecA filament permits DNA polymerase-catalyzed extension of the invading strand in recombination intermediates

Recombination-dependent replication is an essential housekeeping function in prokaryotes and eukaryotes, serving, for example, to restart DNA replication after the repair of a double-strand break. Little is known about the interplay between the recombination and replication machinery when recombination intermediates are used as substrates for DNA replication. We show here that recombination intermediates formed between linear duplex and supercoiled plasmid DNAs are substrates for a generalized strand displacement DNA synthesis reaction in which the 3'-OH of the invading strand in the recombination intermediate is used as a primer. DNA synthesis is driven by negative superhelicity and is inhibited if disassembly of the RecA filament is prevented. Thus, assembly and disassembly of RecA filaments in the same direction facilitates filament clearance from the 3'-end of the invading strand, allowing DNA synthesis to occur from recombination intermediates.