Recombinational DNA Secondary article Repair in Bacteria: Article Contents . Introduction Postreplication . What Leads to Recombinational DNA Repair? . Proteins Involved in Recombinational DNA Repair Kevin P Rice, University of Wisconsin-Madison, Madison, Wisconsin, USA . Pathways . Replication Restart Associated with the SOS Response Michael M Cox, University of Wisconsin-Madison, Madison, Wisconsin, USA . Summary Recombinational DNA repair represents the primary function for homologous DNA recombination in bacteria. Most of this repair occurs at replication forks that are stalled at sites of DNA damage. Introduction fork (including DNA polymerase III and the DnaG and Deoxyribonucleic acid (DNA) damage is a common DnaB proteins) processes in each direction from that occurrence in all cells. A bacterial cell growing in an origin. The forks eventually meet, yielding two identical aerobic environment will suffer 3000–5000 DNA lesions copies ofthe bacterium’s genetic material. per cell per generation (most ofthem oxidative in origin). Recombinational DNA repair ensues whenever a Most of this damage is faithfully repaired by specialized replication fork is halted by DNA damage prior to normal DNA repair systems; however, replication forks will replication termination. There are at least two major types occasionally encounter unrepaired DNA lesions. Because ofdamage and corresponding pathways fortheir repair DNA polymerase is usually unable to bypass DNA (Figure 1). Ifthe forkencounters a DNA strand break, a damage, the complex breaks down in what is best described double-strand break that separates one branch ofthe fork as an enzymatic train wreck. Under normal cellular growth from the rest results. Alternatively, the fork might conditions, nearly every bacterial replication fork will encounter an unrepaired DNA lesion, leaving the lesion suffer this fate. A stalled replication fork triggers an in a single-strand gap at the stalled fork. In at least some elaborate enzymatic response, ultimately restoring an cases, such stalled forks are cleaved to generate double- active replication fork without introducing a genetic strand breaks. The pathways for reactivation of the mutation. replication forks vary to some degree, depending on the While traditionally thought ofas an important avenue DNA structures presented to the cell. The cellular for the generation of genetic diversity, primarily during recombinational DNA repair system is an adaptable set conjugation, homologous DNA recombination in bacteria ofenzymes that can process whatever DNA structures is now understood to be ofparamount importance to the might exist at a collapsed replication fork. general maintenance ofthe genome. The response to a The link between DNA damage, stalled replication forks stalled replication fork involves homologous genetic and homologous recombination is especially easy to see in recombination, followed by a specialized process for the cells that suffer extensive DNA damage, as may happen origin-independent reinitiation ofreplication. The reacti- upon exposure to ultraviolet light, chemical mutagens or vation ofstalled replication forksis a housekeeping ionizing radiation. In such circumstances, cell growth is activity, and represents the primary function of the halted as DNA replication becomes blocked, and the SOS recombination enzymes and the additional proteins and response is induced. After some period of time, DNA enzymes needed for replication restart (Cox, 1998). synthesis resumes and the bacteria continue to divide. This article focuses on recombinational DNA repair in Resumption ofreplication is a phenomenon that is Escherichia coli. completely dependent upon several genes, including most ofthose associated with homologous recombination. The induction ofthe SOS response requires the single-strand gaps that appear at stalled replication forks, and no SOS What Leads to Recombinational DNA induction is observed unless active replication is occurring in the cell. In cells not undergoing conjugation or Repair? transduction, little recombination is observed in the absence ofboth DNA damage and active replication. Normal DNA replication in E. coli is initiated by recruiting The frequency with which replication fork reactivation several proteins to the origin ofreplication, oriC, on the 4.7 is required under normal aerobic growth conditions has million-bp circular chromosome. An active replication ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1 Recombinational DNA Repair in Bacteria: Postreplication oriC-dependent replication Replication (Gap repair) (Double-strand break repair) DNA lesion DNA nick Replication fork Replication fork demise demise Homologous recombination (Breakage) RecA + RecFOR RecA + RecBCD Resolution Resolution (RuvABC + RecG) (RuvABC or RecG) Origin-independent replication restart Replication restart primosome DNA polymerases II + III Replication Completion of replication Figure 1 Pathways for the reactivation of stalled replication forks in bacteria. The pathways diverge depending on what type of damage is encountered. If an unrepaired lesion is encountered (left path), the lesion is left in a DNA gap. A complementary strand is recruited from the other side of the replication fork, with the aid of the RecA, RecFOR, and probably other proteins. Once recombination intermediates are processed (using the RuvABC and/or RecG activities), replication is reinitiated by a specialized set of replication restart enzymes. If the fork instead encounters a DNA strand break, a double-strand break results (right path). The major repair pathway in this case utilizes the RecBCD enzyme. Processing of recombination intermediates and replication restart ensues, as described above. Some of the recombination intermediates generated in the gap (left) repair path may be cleaved and thus funnelled into the double-strand break repair path, as indicated by the broken red arrow. been difficult to quantify. A variety of studies, mostly certain recombination genes, detailed below, produce up to indirect, have yielded estimates ranging from 15 to 100% of a 50% decrease in viability. Mutations that block the replication forks that initiate at oriC. Bacterial mutants replication restart are nearly inviable, suggesting a high defective in double-strand break repair exhibit unrepaired percentage ofreplication forksstalling at DNA damage. double-strand breaks in 15–20% ofcells. Null mutations in The recombination required for replication fork reactiva- 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Recombinational DNA Repair in Bacteria: Postreplication tion often results in DNA crossovers, which can lead to the formation of contiguous chromosome dimers rather than two monomeric chromosomes once replication is com- pleted. Ifthe site-specific recombination system that resolves chromosomal dimers to monomers (the XerCD system) is not functioning, about 15% of the cells have unresolved chromosomal dimers that almost certainly result from recombinational DNA repair of replication forks (Steiner and Kuempel, 1998). Although more quantitation is needed, all ofthe data reinforcethe general idea ofrecombinational DNA repair as an important housekeeping function in bacterial cells, providing non- mutagenic pathways for the reactivation of replication forks stalled by DNA damage. Proteins Involved in Recombinational DNA Repair The reactivation ofstalled replication forksrequires a wide range ofenzymatic activities. The RecA protein is the central factor in recombinational DNA repair. However, several other proteins are required for recombination, including the single-stranded DNA-binding protein (SSB), Figure 2 The bacterial RecA protein and DNA strand exchange. (a) A the RecBCD proteins, the RecFOR proteins, the RecG portion of a RecA filament is shown, containing 24 RecA monomers. There protein and the RuvABC proteins. Also required is the set are approximately six monomers per turn in the helical filament, and a of proteins needed for replication restart. The functions of single monomer is highlighted in red. The DNA is bound in the filament these proteins are integrated, and sometimes redundant. groove. The image is based on the structural work of Randy Story and The XerCD site-specific recombination system is also Thomas Steitz, using PDB file 1REB. (b) Typical DNA strand exchange reactions ulitized to study RecA protein function in vitro. RecA filaments will required to undo some ofthe potentially deleterious effects form on the single-stranded or gapped DNA circles. DNA pairing and inherent to recombinational DNA repair. strand exchange will then ensue with the double-stranded linear DNA molecule. The four-strand exchange exhibits an absolute requirement for ATP hydrolysis. The DNA substrates are usually derived from bacteriophage RecA protein or plasmid DNAs, and the reactions shown were originally chosen for ease of assay. RecA protein promotes the central steps ofany recombi- national process, including the alignment oftwo homo- ectionally (5’!3’) into a nucleoprotein filament (Figure 2a). logous DNA molecules and the subsequent exchange of This filament represents the active species ofRecA protein DNA strands. The recA gene has been identified in every and has several activities that mimic its presumed in vivo bacterial genome in which it has been sought, and RecA role. RecA monomers within a DNA-bound filament on protein has been isolated from perhaps a dozen bacterial 2 1 ssDNA hydrolyse ATP relatively