CDK targets Sae2 to control DNA-end resection and homologous recombination

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Delta null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.     

Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads approximately 40 kb in either direction from the DSB. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX is required for histone loss, which also depends on INO80, a nucleosome remodelling complex. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage.     

ATM controls meiotic double-strand-break formation

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.     

A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.     

Binding of double-strand breaks in DNA by human Rad52 protein

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.     

Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires resection of 5'-termini to generate 3'-single-strand DNA tails. Key components of this reaction are exonuclease 1 and the bifunctional endo/exonuclease, Mre11 (refs 2-4). Mre11 endonuclease activity is critical when DSB termini are blocked by bound protein--such as by the DNA end-joining complex, topoisomerases or the meiotic transesterase Spo11 (refs 7-13)--but a specific function for the Mre11 3'-5' exonuclease activity has remained elusive. Here we use Saccharomyces cerevisiae to reveal a role for the Mre11 exonuclease during the resection of Spo11-linked 5'-DNA termini in vivo. We show that the residual resection observed in Exo1-mutant cells is dependent on Mre11, and that both exonuclease activities are required for efficient DSB repair. Previous work has indicated that resection traverses unidirectionally. Using a combination of physical assays for 5'-end processing, our results indicate an alternative mechanism involving bidirectional resection. First, Mre11 nicks the strand to be resected up to 300 nucleotides from the 5'-terminus of the DSB--much further away than previously assumed. Second, this nick enables resection in a bidirectional manner, using Exo1 in the 5'-3' direction away from the DSB, and Mre11 in the 3'-5' direction towards the DSB end. Mre11 exonuclease activity also confers resistance to DNA damage in cycling cells, suggesting that Mre11-catalysed resection may be a general feature of various DNA repair pathways.     

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.     

The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction

The BRCA2 tumour suppressor is essential for the error-free repair of double-strand breaks (DSBs) in DNA by homologous recombination. This is mediated by RAD51, which forms a nucleoprotein filament with the 3' overhanging single-stranded DNA (ssDNA) of the resected DSB, searches for a homologous donor sequence, and catalyses strand exchange with the donor DNA. The 3,418-amino-acid BRCA2 contains eight approximately 30-amino-acid BRC repeats that bind RAD51 (refs 5, 6) and a approximately 700-amino-acid DBD domain that binds ssDNA. The isolated BRC and DBD domains have the opposing effects of inhibiting and stimulating recombination, respectively, and the role of BRCA2 in repair has been unclear. Here we show that a full-length BRCA2 homologue (Brh2) stimulates Rad51-mediated recombination at substoichiometric concentrations relative to Rad51. Brh2 recruits Rad51 to DNA and facilitates the nucleation of the filament, which is then elongated by the pool of free Rad51. Brh2 acts preferentially at a junction between double-stranded DNA (dsDNA) and ssDNA, with strict specificity for the 3' overhang polarity of a resected DSB. These results establish a BRCA2 function in RAD51-mediated DSB repair and explain the loss of this repair capacity in BRCA2-associated cancers.     

Endonucleolytic processing of covalent protein-linked DNA double-strand breaks

DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 to initiate meiotic recombination. The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal is unclear. Here we show that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3'-OH. Two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-much earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.     

Human CtIP promotes DNA end resection

In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.     

Functional analysis of the sbcD （dr1921 ） gene of the extremely radioresistant bacterium Deinococcus radiodurans

In eukaryotes, the Mre11-Rad50-Nbs1 (MRN) complex, which resides at the crossroads of DNA repair and checkpoint signaling, rapidly forms prominent foci at damage sites following double-strand break (DSB) induction. This complex carries out the initial processing of the DSB ends. Mutations in the genes that encode components of this complex result in DNA-damage hypersensitivity, genomic in- stability, telomere shortening, and aberrant meiosis. Therefore, the MR proteins are highly conserved during evolution. The bacterial orthologs of Mre11 and Rad50 are the SbcD and SbcC proteins, respec- tively. Deinococcus radiodurans, an extremely radioresistant bacterium, is able to mend hundreds of radiation-induced DSBs. The SbcD and SbcC proteins were identified as the products of the Dr1921 and Dr1922 genes. Disruption of the sbcD gene, by direct reverse-orientation insertional mutagenesis tech- nology, remarkably increases the cells’ sensitivity to various types of DNA damaging agents, such as ionizing radiation, ultraviolet irradiation, hydrogen peroxide, and mitomycin C. We also provide evidence that the drSbcD protein plays an important role in both growth and DNA repair in this organ- ism, especially in repair of DSBs generated after cellular exposure to 6000 Gy of IR. These results demonstrate that the drSbcD protein plays an important role in DSBs repair in D. radiodurans.     

Frequent chromosomal translocations induced by DNA double-strand breaks

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.     

ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks

Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.     

Template switching during break-induced replication

DNA double-strand breaks (DSBs) are potentially lethal lesions that arise spontaneously during normal cellular metabolism, as a consequence of environmental genotoxins or radiation, or during programmed recombination processes. Repair of DSBs by homologous recombination generally occurs by gene conversion resulting from transfer of information from an intact donor duplex to both ends of the break site of the broken chromosome. In mitotic cells, gene conversion is rarely associated with reciprocal exchange and thus limits loss of heterozygosity for markers downstream of the site of repair and restricts potentially deleterious chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). BIR from one of the two ends of a DSB would result in loss of heterozygosity, suggesting that BIR is suppressed when DSBs have two ends so that repair occurs by the more conservative gene conversion mechanism. Here we show that BIR can occur by several rounds of strand invasion, DNA synthesis and dissociation. We further show that chromosome rearrangements can occur during BIR if dissociation and reinvasion occur within dispersed repeated sequences. This dynamic process could function to promote gene conversion by capture of the displaced invading strand at two-ended DSBs to prevent BIR.     

Rag mutations reveal robust alternative end joining

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.     

PML contributes to p53-independent p21 up-regulation in gamma-irradiation induced DNA damage responses

The cyclin-dependent kinase inhibitor p21 WAF1/Cip1 is a critical cell cycle regulator which translocates into the nucleus to participate in DNA repair during DNA damage responses. In the present study, we showed that the tumor suppressor, promyelocytic leukemia protein (PML) contributes to the up-regulation of p21 in a p53-independent pathway. Knock-down of PML in p53-null H1299 and HCT 116 (p53 –/– ) tumor cells by specific siRNA resulted in down-regulation of p21 protein expression, inhibition of -irradi...     

Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.     

The mechanism of OTUB1-mediated inhibition of ubiquitination

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13～Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13～Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13～Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13～Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13～Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13～Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.     

EGFR信号转导机制及靶向治疗

综述了EGFR基本结构特征及其介导细胞信号转导的机制,论述了基于EGFR靶向治疗的机理及研究现状。指出,EGFR是最早被发现并研究的RTK家族成员,其蛋白结构分成胞外域、跨膜区与胞内域三部分。EGFR介导细胞信号转导的核心机制是配体EGF与EGFR胞外域结合,通过变构作用与二聚化作用,使胞内域通过反式激活完成对受体末端酪氨酸残基的磷酸化,进而招募下游信号因子完成细胞信号转导过程。质膜结构与组成、EGFR跨膜区的组成及胞外域的有无、EGFR的内吞及泛素依赖性降解过程都调控EGFR细胞信号转导过程。阻断EGFR信号通路可抑制表皮肿瘤细胞成长。EGFR已经成为表皮肿瘤治疗的重要靶点。