Michael M. Cox

The importance of repairing stalled replication forks

The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.

Deinococcus radiodurans — the consummate survivor

Relatively little is known about the biochemical basis of the capacity of Deinococcus radiodurans to endure the genetic insult that results from exposure to ionizing radiation and can include hundreds of DNA double-strand breaks. However, recent reports indicate that this species compensates for extensive DNA damage through adaptations that allow cells to avoid the potentially detrimental effects of DNA strand breaks. It seems that D. radiodurans uses mechanisms that limit DNA degradation and that restrict the diffusion of DNA fragments that are produced following irradiation, to preserve genetic integrity. These mechanisms also increase the efficiency of the DNA-repair proteins.

SSB as an organizer/mobilizer of genome maintenance complexes

When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems. Keywords

Regulation of Bacterial RecA Protein Function

ABSTRACT The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.

Crystal structure of a Flp recombinase-Holliday junction complex: assembly of an active oligomer by helix swapping.

Abstract The crystal structure of a Flp recombinase tetramer bound to a Holliday junction intermediate has been determined at 2.65 A resolution. Only one of Flps two domains, containing the active site, is structurally related to other λ integrase family site-specific recombinases, such as Cre. The Flp active site differs, however, in that the helix containing the nucleophilic tyrosine is domain swapped, such that it cuts its DNA target in trans. The Flp tetramer displays pseudo four-fold symmetry matching that of the square planar Holliday junction substrate. This tetramer is stabilized by additional novel trans interactions among monomers. The structure illustrates how mechanistic unity is maintained on a chemical level while allowing for substantial variation on the structural level within a family of enzymes.

Motoring along with the bacterial RecA protein

The recombinases of the RecA family are often viewed only as DNA-pairing proteins — they bind to one DNA segment, align it with homologous sequences in another DNA segment, promote an exchange of DNA strands and then dissociate. To a first approximation, this description seems to fit the eukaryotic (Rad51 and Dmc1) and archaeal (RadA) RecA homologues. However, the bacterial RecA protein does much more, coupling ATP hydrolysis with DNA-strand exchange in a manner that greatly expands its repertoire of activities. This article explores the protein activities and experimental results that have identified RecA as a motor protein.

Recombinational DNA Repair: The RecF and RecR Proteins Limit the Extension of RecA Filaments beyond Single-Strand DNA Gaps

In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair.

Recombinational DNA Repair in Bacteria and the RecA Protein

In bacteria, the major function of homologous genetic recombination is recombinational DNA repair. This is not a process reserved only for rare double-strand breaks caused by ionizing radiation, nor is it limited to situations in which the SOS response has been induced. Recombinational DNA repair in bacteria is closely tied to the cellular replication systems, and it functions to repair damage at stalled replication forks, Studies with a variety of rec mutants, carried out under normal aerobic growth conditions, consistently suggest that at least 10-30% of all replication forks originating at the bacterial origin of replication are halted by DNA damage and must undergo recombinational DNA repair. The actual frequency may be much higher. Recombinational DNA repair is both the most complex and the least understood of bacterial DNA repair processes. When replication forks encounter a DNA lesion or strand break, repair is mediated by an adaptable set of pathways encompassing most of the enzymes involved in DNA metabolism. There are five separate enzymatic processes involved in these repair events: (1) The replication fork assembled at OriC stalls and/or collapses when encountering DNA damage. (2) Recombination enzymes provide a complementary strand for a lesion isolated in a single-strand gap, or reconstruct a branched DNA at the site of a double-strand break. (3) The phi X174-type primosome (or repair primosome) functions in the origin-independent reassembly of the replication fork. (4) The XerCD site-specific recombination system resolves the dimeric chromosomes that are the inevitable by-product of frequent recombination associated with recombinational DNA repair. (5) DNA excision repair and other repair systems eliminate lesions left behind in double-stranded DNA. The RecA protein plays a central role in the recombination phase of the process. Among its many activities, RecA protein is a motor protein, coupling the hydrolysis of ATP to the movement of DNA branches.

RecA protein promotes the regression of stalled replication forks in vitro

Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria.

A broadening view of recombinational DNA repair in bacteria

Recombinational DNA repair is both the most complex and least understood of DNA repair pathways. In bacterial cells grown under normal laboratory conditions (without a DNA damaging treatment other than an aerobic environment), a substantial number (10–50%) of the replication forks originating at oriC encounter a DNA lesion or strand break. When this occurs, repair is mediated by an elaborate set of recombinational DNA repair pathways which encompass most of the enzymes involved in DNA metabolism. Four steps are discussed: (i) The replication fork stalls and/or collapses. (ii) Recombination enzymes are recruited to the location of the lesion, and function with nearly perfect efficiency and fidelity. (iii) Additional enzymatic systems, including the φX174‐type primosome (or repair primosome), then function in the origin‐independent reassembly of the replication fork. (iv) Frequent recombination associated with recombinational DNA repair leads to the formation of dimeric chromosomes, which are monomerized by the XerCD site‐specific recombination system.