Review
Homologous recombinational repair of DNA ensures mammalian chromosome stability

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Abstract

The process of homologous recombinational repair (HRR) is a major DNA repair pathway that acts on double-strand breaks and interstrand crosslinks, and probably to a lesser extent on other kinds of DNA damage. HRR provides a mechanism for the error-free removal of damage present in DNA that has replicated (S and G2 phases). Thus, HRR acts in a critical way, in coordination with the S and G2 checkpoint machinery, to eliminate chromosomal breaks before the cell division occurs. Many of the human HRR genes, including five Rad51 paralogs, have been identified, and knockout mutants for most of these genes are available in chicken DT40 cells. In the mouse, most of the knockout mutations cause embryonic lethality. The Brca1 and Brca2 breast cancer susceptibility genes appear to be intimately involved in HRR, but the mechanistic basis is unknown. Biochemical studies with purified proteins and cell extracts, combined with cytological studies of nuclear foci, have begun to establish an outline of the steps in mammalian HRR. This pathway is subject to complex regulatory controls from the checkpoint machinery and other processes, and there is increasing evidence that loss of HRR gene function can contribute to tumor development. This review article is meant to be an update of our previous review [Biochimie 81 (1999) 87].

Introduction

This review is a sequel to the article we wrote less than 2 years ago [1] in an area of DNA repair and cancer biology that is developing very rapidly. Here we emphasize current findings for the proteins that participate directly in homologous recombinational repair (HRR), its regulation within the framework of cell cycle checkpoints, and its involvement in human cancer. We cite previously referenced publications only in some instances to reiterate certain points. Other recent reviews emphasize various aspects of homologous recombination in the context of double-strand break (DSB) repair in mammalian cells [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13] or yeast [14], [15].

Genomic instability can be viewed from various perspectives, but here we are particularly concerned with the recombinational repair processes, which are responsible for DNA-strand integrity at the level of the whole chromosome. A distinguishing feature between normal diploid cells and most cancer cells is the ability to avoid chromosomal rearrangements, i.e. translocations, deletions, duplications, and inversions. These abnormalities typically associated with cancer cells arise when the DSB repair systems have failed, either directly, or as a consequence of defects in the coupling of repair with modulation in cell cycle progression. DSBs in vertebrates are repaired by both non-homologous end joining (NHEJ) and HRR [5], [12], [16], [17], [18]. Until recently, NHEJ was thought to be the primary mechanism in mammalian cells for repairing DSBs, which are the principal lethal lesion produced by ionizing radiation (IR). This view was supported by the fact that rodent-cell mutants for several genes (Ku70, Ku86, DNA-PKcs, LIG4, and XRCC4) participating in NHEJ show high sensitivity (up to ∼6-fold) to cell killing and induction of chromosomal aberrations in response to IR [19]. These mutants show defective rejoining of IR-induced DSBs although their deficiencies are incomplete [19].

Only recently were mutants derived from screening procedures identified [20], [21] as being defective in the HRR pathway, which is mediated by the highly conserved Rad51 strand transferase (see Fig. 1 for participating proteins). In particular, the xrcc2 and xrcc3 hamster lines have significant (∼2-fold) IR sensitivity, and they show no detectable defects in IR-induced DSB repair in electrophoretic assays employing supra-lethal doses (>10 Gy). Nevertheless, as indicated by chromosomal aberrations, the xrcc2 and xrcc3 mutants experience high levels of spontaneous and IR-induced DSBs [1], [22]. Recent studies employing enzymatically produced site-specific DSBs in introduced sequences revealed gross defects in both these mutants for the removal of these DSBs by HRR (discussed in Section 2.1) [23], [24].

There is now much evidence that DNA replication generates DSBs that are efficiently repaired through HRR between sister chromatids [15], [25]. A single-strand break located immediately in front of a replication fork could be converted to a DSB. Thus, the high level of spontaneous chromosomal aberrations in the xrcc2 and xrcc3 mutants may be attributed to a partial deficiency in HRR normally occurring between chromatids that have replicated. A complete deficiency, for example through loss of Rad51 function, leads to loss of cell viability [25], [26], [27]. The evidence for a role of NHEJ in eliminating spontaneous DSBs has been paradoxical. CHO ku86/xrcc5 and chicken DT40 ku70 mutants show little or no increase in chromosomal aberrations [16], [28]. In contrast, ku86 knockout mice show vastly elevated levels of chromosomal aberrations in cultured embryonic and adult-skin fibroblasts (56 and 83% of metaphase cells, respectively) [29], [30]. Curiously, the extremely poor growth of ku86-/- MEFs [30] appears inconsistent with the fact that these cells were derived from embryos that were able to complete development. The xrcc4-/- MEFs (mouse embryo fibroblasts) also have very high levels of chromosomal aberrations (∼50% of cells) [31].

Both NHEJ [30], [32] and HRR (see 2.2 Properties of knockout mutations of HRR genes in the mouse, 6 Involvement of HRR pathway in human cancer) likely contribute to the suppression of tumorigenesis. During the development of a malignant phenotype, pre-cancerous cells that are compromised for HRR would have to rely more on NHEJ in S and G2 phases to remove DSBs. Because NHEJ is inherently error-prone, this shift would likely generate more chromosomal rearrangements such as translocations [33], [34], as well as small deletions and insertions [17]. One can speculate that improperly regulated HRR may promote the loss of heterozygosity (LOH, and unmasking of oncogenic mutations) that is often seen in tumor cells [35]. The repair of DSBs through interaction of homologous chromosomes is much less efficient than between chromatids, which may protect cells from the effects of LOH [12].

Sister-chromatid exchanges (SCEs), which are measured cytologically by differential staining of sister chromatids, are induced by many DNA-damaging agents and have long been speculated to arise from homologous recombination although other models based on NHEJ have also been proposed [36]. Recent studies support a mechanism of homologous recombination in these exchange events. Specifically, spontaneous and mitomycin C-induced SCE levels were significantly reduced in chicken or mouse mutants having diminished levels of Rad51 or Rad54 proteins but were normal in ku70 mutant cells [37], [38]. Thus, a substantial fraction of SCEs likely arise from reciprocal exchange occurring through the resolution of Holliday junctions (see Fig. 1 legend). However, the emerging picture suggests that very few DSBs present in replicated DNA get repaired through a mechanism that results in SCE. Studies utilizing I-SceI endonuclease to create DSBs in direct-repeat substrates indicate that sister-chromatid gene conversion is the predominant pathway of repair and that reciprocal exchange rarely occurs (<3% of repair events) [39]. These findings help explain why IR [40] and restriction enzymes [41], [42] are weak inducers of SCEs.

Section snippets

Roles of Rad51 paralogs and Rad54 in the repair of DSBs by HRR

The core reactions of homologous pairing, strand-transfer, and strand exchange or strand annealing (see Fig. 1, Fig. 2, right box) likely involve the trimeric single-strand binding protein, RPA, the human homologs of Saccharomyces cerevisiae Rad51, Rad52, Rad54, and five proteins that we have designated Rad51 “paralogs”. Paralogs are genes that arose through duplication of an ancestral gene and acquired new functions. In this case the paralogs show a high degree of evolutionary divergence from

HsRad51 protein

The eukaryotic Rad51 protein has been shown to be the structural and functional homolog of the prokaryotic RecA strand-transfer protein (reviewed in [1], [67], [68], [69]). Both RecA and Rad51 proteins contain Walker motifs and bind and hydrolyze ATP while forming a filament on single-stranded DNA and performing strand transfer. ATP (actually ATP-Mg2+) frequently plays a dual role: its binding acts as an allosteric effector, and its hydrolysis serves as an energy source. A rad51 mutation in the

HsRad51 nuclear foci

The mammalian Rad51 protein is found in nuclear foci during S and G2 phases of proliferating cells, and IR exposure increases the number of foci (reviewed in [1]). The biological significance of these foci is still not well understood, but several recent studies have been enlightening. Rad51 foci were shown to include single-strand DNA binding protein and to co-localize to ssDNA following treatment with γ-rays, MMC, or etoposide [105]. Each of these DNA damaging agents is known to induce DSBs,

Kinase cascades controlled by Atm and Atr

Cell cycle “checkpoints” are regulatory processes that ensure orderly progression of events during the cell cycle, e.g. the dependence of initiation of mitosis on the completion of DNA replication [131], [132]. When cells experience DNA damage or chemically-imposed inhibition of DNA replication, they respond by activating checkpoint pathways that down-regulate cell cycle progression and coordinate DNA repair processes, as recently reviewed in the context of DSB repair pathways [8].

Involvement of HRR pathway in human cancer

Several recent studies of the genes encoding the HRR enzymatic machinery (see yellow boxes in Fig. 2) point toward the involvement of this pathway in cancer causation. In a study of 127 breast carcinomas [204], LOH detected by polymorphism markers was seen at the regions of Rad51 at 15q15.1 (32% of tumors), Rad52 at 12p13 (16%), and Rad54 at 1p32 (20%). In this study, the BRCA1 (17q21) and BRCA2 (13q12–13) regions had even higher LOH of 49% and 44%, respectively. Moreover, the number of cases

Concluding remarks

The role of HRR in maintaining chromosome stability probably depends heavily on the integrity of the G2/M checkpoint, which helps ensure that DSBs remaining after DNA replication, or produced in G2, are repaired in an error-free manner. When this checkpoint is compromised, there is a much greater likelihood that cells will enter mitosis with chromosome breaks. If the HRR machinery is defective during the period of G2 arrest, the cell must then rely on error-prone NHEJ. This situation will

Acknowledgements

We thank Maria Jasin, Robert Tebbs, and David Wilson III for valuable comments on the manuscript. This work was prepared under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract no. W-7405-ENG-48 and was supported by a National Institutes of Health Grant GM30990 to DS.

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