A role for homologous recombination proteins in cell cycle regulation

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.     

The role of RAD6 in recombinational repair,checkpoints and meiosis via histone modification

The Rad6 ubiquitin-conjugating enzyme in Saccharomyces cerevisiae is known to interact with three separate ubiquitin ligase proteins (Ubr1, Rad18, and Bre1) specific to different targets. The Rad6/Rad18 complex is central to translesion synthesis and the family of DNA transactions known as post-replication repair (PRR). A less well-known aspect of Rad6-mediated DNA repair, however, involves its function with Bre1 in mono-ubiquitinating the histone H2B residue lysine 123. Here, we review how this ubiquitination impacts histone H3 methylation, and how this in turn impacts the DNA damage response. In S. cerevisiae this pathway is required for checkpoint activation in G1, and contributes to DNA repair via the homologous recombination pathway (HRR) in G2 cells. Thus, RAD6 clearly plays a role in HRR in addition to its central role in PRR. We also summarize what is known about related repair pathways in other eukaryotes, including mammals. Recent literature emphasizes the role of methylated histones in S. cerevisiae, Schizosaccharomyces pombe and mammals in attracting the related DNA damage checkpoint proteins Rad9, Crb2 and 53BP1, respectively, to chromatin at the sites of DNA double-strand breaks. However, the specific histone modification pathways involved diverge in these different eukaryotes.     

Localization of checkpoint and repair proteins in eukaryotes

In eukaryotes, the cellular response to DNA damage depends on the type of DNA structure being recognized by the checkpoint and repair machinery. DNA ends and single-stranded DNA are hallmarks of double-strand breaks and replication stress. These two structures are recognized by distinct sets of proteins, which are reorganized into a focal assembly at the lesion. Moreover, the composition of these foci is coordinated with cell cycle progression, reflecting the favoring of end-joining in the G1 phase and homologous recombination in S and G2. The assembly of proteins at sites of DNA damage is largely controlled by a network of protein-protein interactions, with the Mre11 complex initiating assembly at DNA ends and replication protein A directing recruitment to single-stranded DNA. This review summarizes current knowledge on the cellular organization of DSB repair and checkpoint proteins focusing on budding yeast and mammalian cells.     

Differential regulation of the cellular response to DNA double-strand breaks in G1

Double-strand breaks (DSBs) are potentially lethal DNA lesions that can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that DSBs induced by ionizing radiation (IR) are efficiently processed for HR and bound by Rfa1 during G1, while endonuclease-induced breaks are recognized by Rfa1 only after the cell enters S phase. This difference is dependent on the DNA end-binding Yku70/Yku80 complex. Cell-cycle regulation is also observed in the DNA damage checkpoint response. Specifically, the 9-1-1 complex is required in G1 cells to recruit the Ddc2 checkpoint protein to damaged DNA, while, upon entry into S phase, the cyclin-dependent kinase Cdc28 and the 9-1-1 complex both serve to recruit Ddc2 to foci. Together, these results demonstrate that the DNA repair machinery distinguishes between different types of damage in G1, which translates into different modes of checkpoint activation in G1 and S/G2 cells.     

Homology-directed repair is required for the development of radioresistance during S phase: interplay between double-strand break repair and checkpoint response

The S-phase-dependent radioresistance to killing uniformly seen in eukaryotic cells is absent in radiosensitive mutants with defects in genes involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination (homologous recombination repair: HRR). This implicates, for the first time, a concrete DNA repair process in the radiosensitivity of a specific cell cycle phase. The cell cycle-dependent fluctuations in radiosensitivity reflect a fundamental and well-documented radiobiological phenomenon that still awaits a detailed molecular characterization. The underlying mechanisms are likely to combine aspects of DNA repair and cell cycle regulation. Advances in both fields allow a first dissection in the cell cycle of the molecular interplay between DSB repair and DNA damage checkpoint response and its contribution to cell survival. Here we review the available literature on the topic, speculate on the ramifications of this information for our understanding of cellular responses to DNA damage, and discuss future directions in research. An effort is made to integrate relevant phenomena of radiation action, such as low-dose radiosensitivity and the G(2) assay in this scheme.     

Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint

Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.     

The contribution of homologous recombination in preserving genome integrity in mammalian cells

Although it is clear that mammalian somatic cells possess the enzymatic machinery to perform homologous recombination of DNA molecules, the importance of this process in mitigating DNA damage has been uncertain. An initial genetic framework for studying homologous recombinational repair (HRR) has come from identifying relevant genes by homology or by their ability to correct mutants whose phenotypes are suggestive of recombinational defects. While yeast has been an invaluable guide, higher eukaryotes diverge in the details and complexity of HRR. For eliminating DSBs, HRR and end-joining pathways share the burden, with HRR contributing critically during S and G2 phases. It is likely that the removal of interstrand cross-links is absolutely dependent on efficient HRR, as suggested by the extraordinary sensitivity of the ercc1, xpf/ercc4, xrcc2, and xrcc3 mutants to cross-linking chemicals. Similarly, chromosome stability in untreated cells requires intact HRR, which may eliminate DSBs arising during DNA replication and thereby prevent chromosome aberrations. Complex regulation of HRR by cell cycle checkpoint and surveillance functions is suggested not only by direct interactions between human Rad51 and p53, c-Abl, and BRCA2, but also by very high recombination rates in p53-deficient cells.     

Caffeine could not efficiently sensitize homologous recombination repair-deficient cells to ionizing radiation-induced killing

Caffeine inhibits ATM and ATR, two important checkpoint regulators, abolishes ionizing radiation-induced checkpoint response, and radiosensitizes cells. Radiation-induced DNA double-strand breaks (DSBs) are repaired by two major processes, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). It remains unclear which repair process, HRR or NHEJ, is affected when the checkpoint responses are abolished by caffeine. In this study we observed the effect of caffeine on gene-targeted DT40 chicken lymphoblast cells. We show that caffeine efficiently abolishes S- and G(2)-phase checkpoint responses after irradiation in all cell lines tested and greatly radiosensitizes wild-type and ATM(-/-) cells, the partially checkpoint-deficient cells. However, caffeine has a much smaller radiosensitizing effect on RAD54(-/-) cells and has no effect on RAD51-deficient cells. RAD51 and RAD54 are the important factors for HRR. Our results indicate that the checkpoint responses abolished by caffeine (S and G(2)) mainly affect HRR, which results in cell radiosensitization.     

Never-in-mitosis related Kinase 1 functions in DNA damage response and checkpoint control

Nek1, the first mammalian ortholog of the fungal protein kinase never in mitosis A, is involved early in the DNA damage sensing/repair pathway after ionizing radiation. Here we extend this finding by showing that Nek1 localizes to nuclear foci of DNA damage in response to many different types of damage in addition to IR. Untransformed cells established from kat2J/Nek1 -/- mice fail to arrest properly at G1/S and M-phase checkpoints in response to DNA damage. G1-S-phase checkpoint control can be rescued by ectopically overexpressing wild-type Nek1. In Nek1-/- murine cells and in human cells with Nek1 expression silenced by siRNA, the checkpoint kinases Chk1 and Chk2 fail to be activated properly in response to ionizing or UV radiation. In cells without functional Nek1, DNA is not repaired properly, double-stranded DNA breaks persist long after low dose IR, and excessive numbers of chromosome breaks are observed. These data show that Nek1 is important for efficient DNA damage checkpoint control and for proper DNA damage repair.     

Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.     

DNA repair and chromosomal alterations

All mutagenic agents induce lesions in the cellular DNA and they are repaired efficiently by different repair mechanisms. Un-repaired and mis-repaired lesions lead to chromosomal aberrations (CAs). Depending upon the mutagenic agents involved, different DNA repair pathways, such as nucleotide excision repair (NER), base excision repair (BER), non-homologous end joining (NHEJ), homologous recombination repair (HRR), cross-link repair (FANC), single strand annealing (SSA) etc., are operative. Following ionising radiation, DNA double strand breaks (DSBs, which are considered to be the most important leasion leading to observed biological effects) are repaired either by NHEJ and/or HRR. We have investigated the relative role of these two repair pathways leading to chromosomal aberrations using Chinese hamster ovary (CHO) mutant cells deficient in one of these two repair pathwatys. NHEJ operates both in G1 and G2 phases of the cell cycle, wheras HHR operates mainly in S and G2 phases of the cell cycle. In NHEJ-deficient mutant cells irradiated in G1, un-repaired double strand breaks reaching S phase are repaired (unexpectedly with a large mis-repair component) by HRR. In HRR-deficient mutant cells, un-repaired DSBs reaching S phase are repaired by NHEJ (unexpectedly with a low mis-repair component) as evidenced by the frequencies of chromatid type aberrations. Employing a similar approach, following treatment with benzo(alpha)pyrene-7,8diol-9,10epoxide (BPDE), the active metabolite of benzo(alpha)pyrene, NER and HRR seem to be the most important repair pathways protecting against chromosomal damage induced by this agent. In the case of acetaldehyde, (primary metabolite of alcohol in vivo) a DNA cross-linking agent, HRR and FANC pathways are important for protection against damage induced by this agent. Irrespective of the type of DNA lesions induced, ultimately they have to be converted to DSBs in order to give rise to CA. Therefore, both NHEJ and HRR are also involved to some extent in the origin of CA following treatment with S-dependent agents.The relative importance of different repair pathways in bestowing protection against DNA damage leading to chromosomal alterations is discussed.     

Involvement of Brca1 in S-phase and G(2)-phase checkpoints after ionizing irradiation

Cell cycle arrests in the G(1), S, and G(2) phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation. Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase checkpoint, but whether Nbs1 has a definitive role in the G(2)/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G(2) arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G(2)/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1 in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G(2)/M checkpoint.     

Brca1 in immunoglobulin gene conversion and somatic hypermutation

Defects in Brca1 confer susceptibility to breast cancer and genomic instability indicative of aberrant repair of DNA breaks. Brca1 was previously implicated in the homologous recombination pathway via effects on the assembly of recombinase Rad51. Activation-induced cytidine deaminase (AID) deaminates C to U in B lymphocyte immunoglobulin (Ig) DNA to initiate programmed DNA breaks. Subsequent uracil-glycosylase mediated U removal, and perhaps further processing, leads to four known classes of mutation: Ig class switch recombination that results in a region-specific genomic deletion, Ig somatic hypermutation that introduces point mutations in Ig V-regions, Ig gene conversion in vertebrates that possess Ig pseudo-V genes, and translocations common to B cell lymphomas. We tested the involvement of Brca1 in AID-dependent Ig diversification in chicken DT40 cells. The DT40 cell line diversifies IgVlambda mainly by gene conversion, and less so by point mutation. Brca1-deficiency caused a shift in Vlambda diversification, significantly reducing the proportion of gene conversions relative to point mutations. Thus, Brca1 regulates AID-dependent DNA lesion repair. Interestingly, while Brca1 is required to recruit ubiquitinated FancD2 to DNA damage, the phenotype of Brca1-deficient DT40 differs from the one of FancD2-deficient DT40, in which both gene conversion and non-templated mutations are impaired.     

Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways

A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis.     

The Ku-dependent non-homologous end-joining but not other repair pathway is inhibited by high linear energy transfer ionizing radiation

Ionizing radiation (IR) induced DNA double strand breaks (DSBs) are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) in mammalian cells. The NHEJ repair includes a Ku-dependent main pathway and a PARP-1-dependent complementary pathway. Compared with low linear energy transfer (LET) IR (X or gamma ray) at the same doses, high LET IR (high-charge particles) induces more cell death because of ineffective DNA repair. However, it remains unclear whether high LET IR inhibits all repair or specifically one repair pathway. By combining the assays of clonogenic survival, G2M checkpoint and gammaH2AX in the cell lines with deficiencies in different repair genes, we show here that high LET IR inhibits only the Ku-dependent main NHEJ pathway and does not inhibit either the HRR pathway or the PARP-1-dependent complementary NHEJ pathway. In addition, by developing an assay to detect small fragments of DSB (<400 bp) and by detecting the binding abilities of purified Ku and PARP to different sized dsDNA, we present a possible link for explaining the phenotypes. When compared with low LET IR at the same dose, high LET IR might induce similar yields of DNA DSBs in total but it might induce more small fragments of DNA DSBs (<40 base pairs) that prevent Ku binding efficiently to two ends of one DSB fragment at the same time, thus delaying Ku-dependent repair.     

Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

A DNA damage response pathway controlled by Tel1 and the Mre11 complex.

We define a DNA damage checkpoint pathway in S. cerevisiae governed by the ATM homolog Tel1 and the Mre11 complex. In mitotic cells, the Tel1-Mre11 complex pathway triggers Rad53 activation and its interaction with Rad9, whereas in meiosis it acts via Rad9 and the Rad53 paralog Mre4/Mek1. Activation of the Tel1-Mre11 complex pathway checkpoint functions appears to depend upon the Mre11 complex as a damage sensor and, at least in meiotic cells, to depend on unprocessed DNA double-strand breaks (DSBs). The DSB repair functions of the Mre11 complex are enhanced by the pathway, suggesting that the complex both initiates and is regulated by the Tel1-dependent DSB signal. These findings demonstrate that the diverse functions of the Mre11 complex in the cellular DNA damage response are conserved in mammals and yeast.