Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae

The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.     

Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.

We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle. Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions. In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1). Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase. Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates. Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.     

Condensin recruitment to chromatin is inhibited by Chk2 kinase in response to DNA damage

The DNA damage checkpoint, when activated in response to genotoxic damage during S phase, arrests cells in G2 phase of the cell cycle. ATM, ATR, Chk1 and Chk2 kinases are the main effectors of this checkpoint pathway. The checkpoint kinases prevent the onset of mitosis by eliciting well characterized inhibitory phosphorylation of Cdk1. Since Cdk1 is required for the recruitment of condensin, it is thought that upon DNA damage the checkpoint also indirectly blocks chromosome condensation via Cdk1 inhibition. Here we report that the G2 damage checkpoint prevents stable recruitment of the chromosome-packaging-machinery components condensin complex I and II onto the chromatin even in the presence of an active Cdk1. DNA damage-induced inhibition of condensin subunit recruitment is mediated specifically by the Chk2 kinase, implying that the condensin complexes are targeted by the checkpoint in response to DNA damage, independently of Cdk1 inactivation. Thus, the G2 checkpoint directly prevents stable recruitment of condensin complexes to actively prevent chromosome compaction during G2 arrest, presumably to ensure efficient repair of the genomic damage.     

The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation

Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.     

Differential role of RB in response to UV and IR damage

The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of cancers and is a critical mediator of DNA damage checkpoints. Despite the critical importance of RB function in tumor suppression, the coordinate impact of RB loss on the response to environmental and therapeutic sources of damage has remained largely unexplored. Here, we utilized a conditional knockout system to ablate RB in adult fibroblasts. This model system enabled us to investigate the temporal role of RB loss on cell cycle checkpoints and DNA damage repair following ultraviolet (UV) and ionizing radiation (IR) damage. We demonstrate that RB loss compromises rapid cell cycle arrest following UV and IR exposure in adult primary cells. Detailed kinetic analysis of the checkpoint response revealed that disruption of the checkpoint is concomitant with RB target gene deregulation, and is not simply a manifestation of chronic RB loss. RB loss had a differential effect upon repair of the major DNA lesions induced by IR and UV. Whereas RB did not affect resolution of DNA double-strand breaks, RB-deficient cells exhibited accelerated repair of pyrimidine pyrimidone photoproducts (6-4 PP). In parallel, this repair was coupled with enhanced expression of specific factors and the behavior of proliferating cell nuclear antigen (PCNA) recruitment to replication and repair foci. Thus, RB loss and target gene deregulation hastens the repair of specific lesions distinct from its ubiquitous role in checkpoint abrogation.     

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.     

The chromatin remodeling factor BRG1 stimulates nucleotide excision repair by facilitating recruitment of XPC to sites of DNA damage

BRG1 is a catalytic subunit of the human SWI/SNF-like BAF chromatin remodeling complexes. Recent findings have shown that inactivation of BRG1 sensitizes mammalian cells to various DNA damaging agents, including ultraviolet (UV) and ionizing radiation. However, it is unclear whether BRG1 facilitates nucleotide excision repair (NER). Here we show that re-expression of BRG1 in cells lacking endogenous BRG1 expression stimulates nucleotide excision repair of UV induced DNA damage. Using a micropore UV radiation technique, we demonstrate that recruitment of the DNA damage recognition protein XPC to sites of UV lesions is significantly disrupted when BRG1 is depleted. Chromatin immunoprecipitation of the endogenous DDB2 protein, which is involved in recruiting XPC to UV-induced CPDs (cyclobutane pyrimidine dimers), shows that elevated levels of BRG1 are associated with DDB2 in chromatin in response to UV radiation. Additionally, we detected slow BRG1 accumulation at sites of UV lesions. Our findings suggest that the chromatin remodeling factor BRG1 is recruited to sites of UV lesions to facilitate NER in human chromatin.     

Rad51 replication fork recruitment is required for DNA damage tolerance

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non‐repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non‐DSB DNA lesions, presumably single‐stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle‐regulated replicative and repair functions.     

UV irradiation causes the loss of viable mitotic recombinants in Schizosaccharomyces pombe cells lacking the G(2)/M DNA damage checkpoint

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants.     

Specific recruitment of human cohesin to laser-induced DNA damage

Cohesin is a conserved multiprotein complex that plays an essential role in sister chromatid cohesion. During interphase, cohesin is required for the establishment of cohesion following DNA replication. Because cohesin mutants resulted in increased sensitivity to DNA damage, a role for cohesin in DNA repair was also suggested. However, it was unclear whether this was due to general perturbation of cohesion or whether cohesin has a specialized role at the damage site. We therefore used a laser microbeam to create DNA damage at discrete sites in the cell nucleus and observed specific in vivo assembly of proteins at these sites by immunofluorescent detection. We observed that human cohesin is recruited to the damage site immediately after damage induction. Analysis of mutant cells revealed that cohesin recruitment to the damage site is dependent on the DNA double-strand break repair factor Mre11/Rad50 but not ATM or Nbs1. Consistently, Mre11/Rad50 and cohesin interact with each other in an interphase-specific manner. This interaction peaks in S/G(2) phase, during which cohesin is recruited to the DNA damage. Our results demonstrate the S/G(2)-specific and Mre11/Rad50-dependent recruitment of human cohesin to DNA damage, suggesting a specialized subfunction for cohesin in cell cycle-specific DNA double strand break repair.     

Shedding Light on the DNA Damage Checkpoint

DNA damage checkpoint genes are required to restrain cell cycle progression during DNA repair and to maintain chromosome stability. Checkpoint mutants are highly sensitive to killing by UV light, so the responses mediated by these genes are likely to be essential for survival during exposure to solar radiation. Yet it is still unclear exactly how checkpoint responses coordinate the cell cycle with DNA repair in the presence of UV lesions. At high doses, the UV response shares features with the ionizing radiation response, such as G1/S and G2/M checkpoints. At lower doses, only a postreplication checkpoint is evident. In this perspective we attempt to reconcile these observations and address their physiological meaning, with an emphasis on insights gained from direct cell-cycle measurements and recent studies in yeast.     

NER initiation factors,DDB2 and XPC,regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites

ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.