Regulation of DNA repair throughout the cell cycle

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.     

In vitro expression levels of cell-cycle checkpoint proteins are associated with cellular DNA repair capacity in peripheral blood lymphocytes: a multivariate analysis

DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint, and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To test this hypothesis, we performed the host-cell reactivation assay for DRC by transfecting stimulated peripheral blood lymphocytes from 120 normal donors with transient expression plasmids damaged by benzo[a]pyrene diol epoxide (BPDE). The same cells were assessed for protein expression induction of eight cell-cycle checkpoint-related genes using the reverse-phase protein lysate microarray assay. In multivariate linear regression analysis adjusting for age, sex, blastogenic rate, and sample storage duration, the association between DRC and expression levels of cell-cycle checkpoint-related proteins induced by BPDE-adducts was statistically significant for p27, CCND1, ATM, and MDM2 (P = 0.00, 0.03, 0.03, and 0.03, respectively), borderline for p73 and p21 (P = 0.07 and 0.09, respectively), but not for p53 and p16 (P = 0.13 and 0.18, respectively). Because the relative expression levels of all these eight proteins were highly correlated, we further performed the principal component analysis and identified ATM as the most important predictor of DRC, followed by MDM2 and p27. Our results provide population-based in vitro evidence demonstrating that cell-cycle checkpoint-related proteins play essential roles in regulating DNA repair, at least in unaffected human peripheral blood lymphocytes. Further studies are warranted to investigate the role of interindividual variation in the expression levels of these proteins in cancer susceptibility.     

Cell-cycle checkpoint kinases: checking in on the cell cycle

In response to DNA damage, cell-cycle checkpoints integrate cell-cycle control with DNA repair. The idea that checkpoint controls are an integral component of normal cell-cycle progression has arisen as a result of studies in Drosophila and mice. In addition, an appreciation that DNA damage arises as a natural consequence of cellular metabolism, including DNA replication itself, has influenced thinking regarding the nature of checkpoint pathways.     

Molecular mechanisms of DNA double-strand break repair

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.     

The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity

DNA damage leads to activation of several mechanisms such as DNA repair and cell-cycle checkpoints. It is evident that these different cellular mechanisms have to be finely co-ordinated. Growing evidence suggests that the Rad9/Rad1/Hus1 cell-cycle checkpoint complex (9-1-1 complex), which is recruited to DNA lesion upon DNA damage, plays a major role in DNA repair. This complex has been shown to interact with and stimulate several proteins involved in long-patch base excision repair. On the other hand, the well-characterised DNA clamp-proliferating cell nuclear antigen (PCNA) also interacts with and stimulates several of these factors. In this work, we compared the effects of the 9-1-1 complex and PCNA on flap endonuclease 1 (Fen1). Our data suggest that PCNA and the 9-1-1 complex can independently bind to and activate Fen1. Finally, acetylation of Fen1 by p300-HAT abolished the stimulatory effect of the 9-1-1 complex but not that of PCNA, suggesting a possible mechanism of regulation of this important repair pathway.     

Abrogation of the CLK-2 checkpoint leads to tolerance to base-excision repair intermediates

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell-cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil-DNA glycosylase UNG-1. Strikingly, abrogation of the clk-2 checkpoint gene rescued lethality and developmental defects, and eliminated cell-cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG-1 and activation of the CLK-2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single-stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK-2 checkpoint in C. elegans.     

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G₂) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant'' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death.     

Checkpoint mediators: relaying signals from DNA strand breaks

The recently identified checkpoint mediator MDC1 facilitates recruitment of DNA repair proteins to damaged sites and establishment of the intra-S-phase cell-cycle checkpoint. Increasing evidence suggests that proteins like MDC1 provide the framework necessary for transducing signals from DNA double-strand breaks.     

Who binds wins: Competition for PCNA rings out cell-cycle changes

PCNA, proliferating cell nuclear antigen, is a pivotal protein in DNA replication, DNA repair and possibly cell-cycle control. The protein has a trimeric ring structure that might slide along duplex DNA and form a platform for association with a variety of proteins, in particular holding the DNA polymerases in close association with their template. This article reviews evidence suggesting that the activity of PCNA in replication and repair is coordinated within the cell cycle by cooperative and competitive interactions with an extensive network of enzymes and regulatory proteins.     

Functional interaction between FOXO3a and ATM regulates DNA damage response

The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2-M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.     

Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes.     

A naturally occurring human RPA subunit homolog does not support DNA replication or cell-cycle progression

Replication Protein A (RPA) is a single-stranded DNA-binding protein essential for DNA replication, repair, recombination and cell-cycle regulation. A human homolog of the RPA2 subunit, called RPA4, was previously identified and shown to be expressed in colon mucosal and placental cells; however, the function of RPA4 was not determined. To examine the function of RPA4 in human cells, we carried out knockdown and replacement studies to determine whether RPA4 can substitute for RPA2 in the cell. Unlike RPA2, exogenous RPA4 expression did not support chromosomal DNA replication and lead to cell-cycle arrest in G2/M. In addition, RPA4 localized to sites of DNA repair and reduced γ-H2AX caused by RPA2 depletion. These studies suggest that RPA4 cannot support cell proliferation but can support processes that maintain the genomic integrity of the cell.     

Effect of cell-cycle position and dose on the kinetics of DNA double-strand breakage repair in X-irradiated Chinese hamster cells

The kinetics of DNA double-strand breakage (d.s.b.) repair in X-irradiated Chinese hamster V79 cells were found to be affected by cell-cycle position. In mitotic cells, the repair kinetics were monophasic with a half-time value of about 32 min, whilst in G1, S, or asynchronous cultures, the kinetics were biphasic with half-time values of around 2.7 and 27 min. The repair of DNA single-strand breakage (s.s.b) was also shown to be slower in mitotic than in interphase cells. The DNA d.s.b. repair system, in both mitotic and interphase cells, showed no evidence of saturation within the X-ray dose range covered. The implications of these findings for the mechanism of DNA d.s.b. repair and for models of ionizing radiation action are discussed.     

Checking on DNA damage in S phase

The precise replication of the genome and the continuous surveillance of its integrity are essential for survival and the avoidance of various diseases. Cells respond to DNA damage by activating a complex network of the so-called checkpoint pathways to delay their cell-cycle progression and repair the defects. In this review we integrate findings on the emerging mechanisms of activation, the signalling pathways and the spatio-temporal organization of the intra-S-phase DNA-damage checkpoint and its impact on the cell-cycle machinery, and discuss its biological significance.