The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice.

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5'-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.

To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks.

Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.

The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.

How do cells sense DNA lesions?

DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets. These targets, in turn, impact a wide range of cellular processes including DNA replication, DNA repair and cell cycle transitions. The importance of the DDR is highlighted by the fact that DDR inactivation is commonly found in cancer and causes many different human diseases. The protein kinases ATM and ATR, as well as their budding yeast orthologs Tel1 and Mec1, act as master regulators of the DDR. The initiating events in the DDR entail both DNA lesion recognition and assembly of protein complexes at the damaged DNA sites. Here, we review what is known about the early steps of the DDR.

Tel1/ATM Signaling to the Checkpoint Contributes to Replicative Senescence in the Absence of Telomerase.

Telomeres progressively shorten at every round of DNA replication in the absence of telomerase. When they become critically short, telomeres trigger replicative senescence by activating a DNA damage response that is governed by the Mec1/ATR and Tel1/ATM protein kinases. While Mec1/ATR is known to block cell division when extended single-stranded DNA (ssDNA) accumulates at eroded telomeres, the molecular mechanism by which Tel1/ATM promotes senescence is still unclear. By characterizing a Tel1-hy184 mutant variant that compensates for the lack of Mec1 functions, we provide evidence that Tel1 promotes senescence by signaling to a Rad9-dependent checkpoint. Tel1-hy184 anticipates senescence onset in telomerase-negative cells, while the lack of Tel1 or the expression of a kinase-defective (kd) Tel1 variant delays it. Both Tel1-hy184 and Tel1-kd do not alter ssDNA generation at telomeric DNA ends. Furthermore, Rad9 and (only partially) Mec1 are responsible for the precocious senescence promoted by Tel1-hy184. This precocious senescence is mainly caused by the F1751I, D1985N, and E2133K amino acid substitutions, which are located in the FRAP-ATM-TRAPP domain of Tel1 and also increase Tel1 binding to DNA ends. Altogether, these results indicate that Tel1 induces replicative senescence by directly signaling dysfunctional telomeres to the checkpoint machinery.

Processing of DNA Double-Strand Breaks by the MRX Complex in a Chromatin Context.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. The cellular response to DSBs is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that has structural and catalytic functions. Furthermore, it is responsible for DSB signaling through the activation of the checkpoint kinase Tel1/ATM. Here, we review functions and regulation of the MRX/MRN complex in DSB processing in a chromatin context, as well as its interplay with Tel1/ATM.

Uncoupling Sae2 Functions in Downregulation of Tel1 and Rad53 Signaling Activities.

The Mre11-Rad50-Xrs2 (MRX) complex acts together with the Sae2 protein to initiate resection of DNA double-strand breaks (DSBs) and to regulate a checkpoint response that couples cell cycle progression with DSB repair. Sae2 supports resistance to DNA damage and downregulates the signaling activities of MRX, Tel1, and Rad53 checkpoint proteins at the sites of damage. How these functions are connected to each other is not known. Here, we describe the separation-of-function sae2-ms mutant that, similar to SAE2 deletion, upregulates MRX and Tel1 signaling activities at DSBs by reducing Mre11 endonuclease activity. However, unlike SAE2 deletion, Sae2-ms causes neither DNA damage sensitivity nor enhanced Rad53 activation, indicating that DNA damage resistance depends mainly on Sae2-mediated Rad53 inhibition. The lack of Sae2, but not the presence of Sae2-ms, impairs long-range resection and increases both Rad9 accumulation at DSBs and Rad53-Rad9 interaction independently of Mre11 nuclease activity. Altogether, these data lead to a model whereby Sae2 plays distinct functions in limiting MRX-Tel1 and Rad9 abundance at DSBs, with the control on Rad9 association playing the major role in supporting DNA damage resistance and in regulating long-range resection and checkpoint activation.

Processing of DNA Ends in the Maintenance of Genome Stability.

DNA double-strand breaks (DSBs) are particularly hazardous lesions as their inappropriate repair can result in chromosome rearrangements, an important driving force of tumorigenesis. DSBs can be repaired by end joining mechanisms or by homologous recombination (HR). HR requires the action of several nucleases that preferentially remove the 5'-terminated strands at both DSB ends in a process called DNA end resection. The same nucleases are also involved in the processing of replication fork structures. Much of our understanding of these pathways has come from studies in the model organism Saccharomyces cerevisiae. Here, we review the current knowledge of the mechanism of resection at DNA DSBs and replication forks.

Alkaline Denaturing Southern Blot Analysis to Monitor Double-Strand Break Processing.

Generation of 3' single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5' strand degradation proceeds beyond restriction sites. After the separation by electrophoresis on alkaline denaturing agarose gel, these ssDNA fragments can be visualized by hybridization with an RNA probe that anneals with the 3'-undegraded DSB strand. This assay allows a direct and comprehensive visualization of DSB end processing.

The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1.

Eukaryotic cells preserve genome integrity upon DNA damage by activating a signaling network that promotes DNA repair and controls cell cycle progression. One of the most severe DNA damage is the DNA double-strand break (DSB), whose 5΄ ends can be nucleolitically resected by multiple nucleases to create 3΄-ended single-stranded DNA tails that trigger DSB repair by homologous recombination. Here, we identify the Saccharomyces cerevisiae RNA binding protein Npl3 as a new player in DSB resection. Npl3 is related to both the metazoan serine-arginine-rich and the heterogeneous nuclear ribonucleo-proteins. NPL3 deletion impairs the generation of long ssDNA tails at the DSB ends, whereas it does not exacerbate the resection defect of exo1Δ cells. Furthermore, either the lack of Npl3 or the inactivation of its RNA-binding domains causes decrease of the exonuclease Exo1 protein levels as well as generation of unusual and extended EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, indicate that Npl3 participates in DSB resection by promoting the proper biogenesis of EXO1 mRNA.

Resection is responsible for loss of transcription around a double-strand break in Saccharomyces cerevisiae.

Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. Here we show that in Saccharomyces cerevisiae a single DSB causes transcriptional inhibition of proximal genes independently of Tel1/ATM and Mec1/ATR. Since the DSB ends undergo nucleolytic degradation (resection) of their 5'-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition. We discovered that resection-defective mutants fail to stop transcription around a DSB, and the extent of this failure correlates with the severity of the resection defect. Furthermore, Rad9 and generation of γH2A reduce this DSB-induced transcriptional inhibition by counteracting DSB resection. Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S. cerevisiae.