DNA damage checkpoint and repair centers

In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into focal assemblies. These foci are highly dynamic giga-dalton structures capable of simultaneously repairing multiple DNA lesions. Moreover, the composition of these repair centers depends on the nature of the DNA lesion and is tightly coordinated with progression of the cell cycle. Components of DNA repair centers are regulated by post-translational modifications such as phosphorylation, ubiquitination and sumoylation. Repair foci progress through four distinct stages: first, DNA damage recognition and binding of DNA ends by the Mre11 complex and Ku70/80; second, end-processing and binding of single-stranded DNA by replication protein A, which recruits checkpoint proteins; third, recombinational repair during S and G(2) phase; and fourth, disassembly of foci and resumption of the cell cycle.     

The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9-dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences.     

Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control

BRCA1 is a tumor suppressor involved in DNA repair and damage-induced checkpoint controls. In response to DNA damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions. However, little is known about the regulation of BRCA1 relocalization following DNA damage. Here we show that mediator of DNA damage checkpoint protein 1 (MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of DNA damage responses that regulates BRCA1 function. MDC1 regulates ataxia-telangiectasia-mutated (ATM)-dependent phosphorylation events at the site of DNA damage. Importantly down-regulation of MDC1 abolishes the relocalization and hyperphosphorylation of BRCA1 following DNA damage, which coincides with defective G(2)/M checkpoint control in response to DNA damage. Taken together these data suggest that MDC1 regulates BRCA1 function in DNA damage checkpoint control.     

DNA replication as a target of the DNA damage checkpoint

Faithful inheritance of the genome from mother to daughter cell requires that it is replicated accurately, in its entirety, exactly once. DNA replication not only has to have high fidelity, but also has to cope with exogenous and endogenous agents that damage DNA during the life cycle of a cell. The DNA damage checkpoint, which monitors and responds to defects in the genome, is critical for the completion of replication. The focus of this review is how DNA replication is regulated by the checkpoint response in the presence of DNA damage and fork stalling agents.     

Regulation of DNA replication by the S-phase DNA damage checkpoint

Cells slow replication in response to DNA damage. This slowing was the first DNA damage checkpoint response discovered and its study led to the discovery of the central checkpoint kinase, Ataxia Telangiectasia Mutated (ATM). Nonetheless, the manner by which the S-phase DNA damage checkpoint slows replication is still unclear. The checkpoint could slow bulk replication by inhibiting replication origin firing or slowing replication fork progression, and both mechanisms appear to be used. However, assays in various systems using different DNA damaging agents have produced conflicting results as to the relative importance of the two mechanisms. Furthermore, although progress has been made in elucidating the mechanism of origin regulation in vertebrates, the mechanism by which forks are slowed remains unknown. We review both past and present efforts towards determining how cells slow replication in response to damage and try to resolve apparent conflicts and discrepancies within the field. We propose that inhibition of origin firing is a global checkpoint mechanism that reduces overall DNA synthesis whenever the checkpoint is activated, whereas slowing of fork progression reflects a local checkpoint mechanism that only affects replisomes as they encounter DNA damage and therefore only affects overall replication rates in cases of high lesion density.     

Assaying the DNA damage checkpoint in fission yeast

Cell cycle checkpoints exist to ensure the proper maintenance and stable inheritance of genomic information. The pathways that insure the faithful execution of these checkpoints are well conserved throughout evolution. In the fission yeast, Schizosaccharomyces pombe, a major cell cycle checkpoint exists that responds to the presence of damaged DNA and prevents this damage from being propagated to future generations. Fission yeast is an ideal system to investigate these pathways because there exist specific techniques that allow one to assay the fidelity of this DNA damage checkpoint pathway.     

DNA损伤检验点调控的分子机制

多种因素可以引起DNA损伤而最终导致基因产生错义突变、缺失或错误重组。为确保遗传准确性,细胞形成了复杂的细胞周期监督机制,即细胞周期检验点。其中DNA损伤检验点由许多检验点相关蛋白组成,可以识别损伤的DNA,经复杂的信号转导途径引发蛋白激酶的级联反应,减慢或阻滞细胞周期进程,从而为细胞修复损伤的DNA赢得时间。     

A DNA damage checkpoint in Escherichia coli

An early attempt to find out if the DNA double helix is actively unwound before being replicated was not conclusive, but it did disclose the existence of a unique moment in the life cycle of Escherichia coli when the cell registers whether or not its DNA is intact. If not, the cell embarks on rapid breakdown of its DNA, like "apoptosis" in eukaryotic cells.     

DdcA antagonizes a bacterial DNA damage checkpoint

Bacteria coordinate DNA replication and cell division, ensuring a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage, a cell cycle checkpoint is activated by expressing a cell division inhibitor. The prevailing model is that activation of the DNA damage response and protease‐mediated degradation of the inhibitor is sufficient to regulate the checkpoint process. Our recent genome‐wide screens identified the gene ddcA as critical for surviving exposure to DNA damage. Similar to the checkpoint recovery proteases, the DNA damage sensitivity resulting from ddcA deletion depends on the checkpoint enforcement protein YneA. Using several genetic approaches, we show that DdcA function is distinct from the checkpoint recovery process. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels and stability, further supporting the conclusion that DdcA regulates YneA independent of proteolysis. Using a functional GFP‐YneA fusion we found that DdcA prevents YneA‐dependent cell elongation independent of YneA localization. Together, our results suggest that DdcA acts by helping to set a threshold of YneA required to establish the cell cycle checkpoint, uncovering a new regulatory step controlling activation of the DNA damage checkpoint in Bacillus subtilis.     

Centrosomal localization of DNA damage checkpoint proteins

During mitosis, the phosphatidylinositol-3 (PI-3) family-related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA-PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA-PKcs, the catalytic subunit of DNA-PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co-immunoprecipitated with Ku70/86, the DNA binding subunits of DNA-PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of alpha-tubulin foci in the nucleus. Interestingly, gamma-tubulin was also present in the nucleus and co-immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of gamma-tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of gamma-tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response.     

DNA damage checkpoint in budding yeast.

Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.     

Activating the DNA damage checkpoint in a developmental context

BACKGROUND: Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, we examined the effect of X-rays on post-blastoderm embryos of Drosophila melanogaster. RESULTS: In Drosophila, DNA damage was previously found to delay anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that included a G2 phase, we found that irradiation delayed the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurred normally after irradiation. The radiation-induced delay of mitosis accompanied the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drove a mitotic cyclin into the nucleus and overcame the delay of mitosis induced by irradiation. CONCLUSIONS: Developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa.     

Expanded CAG repeats activate the DNA damage checkpoint pathway

Trinucleotide repeats (TNRs) are sequences whose expansion causes several genetic diseases and chromosome breakage. We report a novel finding that expanded CAG repeats activate the DNA damage response. Mutations in yeast MEC1, RAD9, or RAD53 genes result in increased rates of fragility of a CAG repeat tract while single or double deletions of RAD17 or RAD24 have only a modest effect on TNR fragility, indicating that signaling down the Rad9 pathway and not the Rad17-Rad24 pathway plays a major role in sensing and repairing CAG-tract breaks. Deletion of CHK1 had no effect on CAG fragility, suggesting that a Chk1-mediated G2 arrest is not required for TNR repair. Absence of Mec1, Ddc2, Rad17, Rad24, or Rad53 also gives rise to increased frequency of CAG repeat contractions, indicating that components of the checkpoint machinery play an active role in the maintenance of both chromosomal integrity and repeat stability at expanded CAG sequences.