Alternative Lengthening of Telomeres through Two Distinct Break-Induced Replication Pathways

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Extensive DNA End Processing by Exo1 and Sgs1 Inhibits Break-Induced Replication

Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination.     

Mechanisms of Dealing with DNA Damage-Induced Replication Problems

During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.     

Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication

Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.Cellular DNA damage response pathways protect and preserve the integrity of the genome. These pathways, which are activated in response to various forms of DNA damage, involve a number of proteins that participate in both DNA repair and cell cycle progression (62). The serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK) are activated in response to distinct types of damage. The ATM pathway is activated primarily by double-stranded DNA breaks (4, 30). DNA-PK acts in conjunction with the DNA ligase IV/XRCC4 complex to mediate the ligation of double-stranded breaks through nonhomologous end joining (34). The ATR pathway can be activated in response to a wide range of genotoxic stresses, such as base or nucleotide excision, double-stranded breaks, or single-stranded breaks. Activation of ATR is generally thought to occur via the recognition of single-stranded tracks of DNA (63). Each of these pathways leads to the phosphorylation and activation of a number of cellular proteins such as the variant histone H2AX, checkpoint kinases 1 and 2 (Chk1 and Chk2), and Nijmegen break syndrome protein 1 (NBS1), among others (62). Signals transmitted by a cascade of phosphorylation events result in cell cycle arrest and the accumulation of repair protein complexes at sites of DNA damage.Upon recognition of a double-stranded DNA break by the cell, H2AX is phosphorylated on an extended C-terminal tail at serine 139 by the phosphatidylinositol 3-kinase (PI3K)-related kinases ATM, ATR, and DNA-PK (9, 41, 44, 58). Considered one of the earliest indications of a double-stranded DNA break, phosphorylated H2AX (γH2AX) acts as a scaffolding protein to which a number of DNA repair factors can dock to facilitate repair of the damaged DNA (36, 42, 53). Areas of phosphorylated H2AX, termed γH2AX foci, are enriched for proteins involved in both homologous recombination and nonhomologous end joining, such as NBS1, BRCA1 (42), and Mdc1 (24, 50).Although adenovirus is able to activate both ATM and ATR pathways (11), adenoviral proteins limit the extent and consequences of signaling through these pathways. The E1B-55K and E4orf6 proteins form an E3 ubiquitin ligase with the cellular proteins Cullin-5, elongins B and C, and Rbx1 (28, 43). This complex targets key cellular proteins involved in cellular response to DNA damage, including p53 (28, 43), Mre11 (51), and DNA ligase IV (3). The E4orf3 gene product targets cellular proteins central to both the cellular DNA damage response and the antiviral response. The E4orf3 protein of species C adenoviruses alters the localization of Mre11/Rad50/NBS1 (MRN) complex members within the nucleus to prevent association with centers of viral DNA replication and to ensure efficient viral DNA replication (17, 18, 52). In addition, these three viral early proteins direct members of the MRN complex (2, 35) and the single-stranded DNA-binding protein 2 (20) to cytoplasmic aggresomes, where these sequestered proteins are effectively inactivated. These viral activities, along with the inactivation of DNA-PK by E4orf3 and E4orf6 gene products (7), appear to prevent recognition of viral genomes by the MRN complex and prevent ligation of these genomes through nonhomologous end joining. In cells infected with a virus with E4 deleted, Mre11 physically binds to viral DNA in an NBS1-dependent manner and may prevent efficient genome replication (37). The overlapping means by which adenovirus disables the MRN complex and prevents DNA damage repair serves to illustrate the importance of this activity for a productive adenovirus infection. However, despite having DNA damage signaling and DNA repair pathways dismantled, adenovirus-infected cells exhibit some characteristic changes associated with DNA damage signaling events, such as the phosphorylation of H2AX (6, 15). Thus, it appears that adenovirus effectively inhibits DNA repair activity but may not fully suppress the early events of DNA damage signaling.The focus of the present study was to elucidate the activation of DNA damage signaling pathways revealed by phosphorylation of the variant histone H2AX during wild-type adenovirus infection and to determine what stage of the virus life cycle leads to this activation. We demonstrate that infected cells respond to viral genome replication with high levels of H2AX phosphorylation throughout the cell nucleus. This phosphorylation event is not localized to viral replication centers and does not appear to be concurrent with cellular double-stranded DNA breaks; rather, H2AX phosphorylation occurs coincident with the bulk of cellular chromatin. H2AX phosphorylation follows viral DNA replication and reaches peak levels after the degradation of the Mre11. In addition, we observed that infected cells can respond to both the presence of incoming viral genomes and genome replication by initiating H2AX phosphorylation.     

有丝分裂期DNA合成（MiDAS）及其介导的端粒复制

DNA复制压力（replication stress，RS）是一个广泛定义DNA复制障碍的术语，通常是指那些能够扰乱复制进程，造成复制叉减慢或停滞的情况。复制压力的过度累积是肿瘤发生和基因组不稳定的主要驱动因素。细胞染色体在复制过程中会不断地遭受来自外源性或内源性复制压力，而端粒及常见脆性位点（common fragile sites，CFSs）是一类对复制压力高度敏感的区域，在复制压力较高的情况下，这些区域往往难以被完全复制。近年的研究发现，有丝分裂期DNA合成（mitotic DNA repair synthesis，MiDAS）区别于S期的复制，可以帮助难以复制的区域在进入有丝分裂期后仍然能够保证复制的进行，因此，MiDAS也被称为“复制的挽救机制”。由于端粒的维持依赖于端粒酶活性及端粒替代性延长机制（alternative lengthening of telomeres，ALT），而具有更多端粒脆性的ALT细胞中端粒-MiDAS表现出高度的活性，因此本文就MiDAS的发生机制及在不同端粒维持机制下难以复制的端粒如何应对复制压力在有丝分裂期完成DNA的合成进行综述。     

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.     

DNA Damage-Induced Phosphorylation of TRF2 Is Required for the Fast Pathway of DNA Double-Strand Break Repair

Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.Telomeres act as protective caps to disguise the chromosome end from being recognized as a DNA double-strand break (DSB) and play other important roles in maintaining genomic integrity (2, 21, 26). Telomere capping dysfunction resulting in genomic instability is likely a major pathway leading to human cancers and other age-related diseases (8, 27).An increasing number of proteins known to play important roles in DNA repair have also been found to be critical for telomere maintenance (6). Specifically, phosphatidylinositol (PI) 3-kinase-like kinase family members, such as ataxia telangiectasia mutated protein kinase (ATM) and the DNA-dependent protein kinase catalytic subunit in mammals, originally known to act in maintaining genomic stability via DNA repair pathways, have been shown to be important in telomere maintenance (1, 4, 7, 9, 10, 16, 25). Previous reports indicate that ATM is required for the DNA damage-induced phosphorylation of two major telomere-associated proteins in mammals, human TRF1 and TRF2 (16, 28). The specific molecular roles played by the DNA damage-induced phosphorylation of TRF1 and TRF2 in telomere maintenance and/or DNA repair are unclear and under active investigation. We previously reported that upon DNA damage, human TRF2 was rapidly and transiently phosphorylated at threonine 188 (T188) (28). Here, we report that X-ray-induced phosphorylation of human TRF2 at T188 plays a functional role in the fast pathway of DNA DSB repair.     

Efficient Initiation of DNA Replication in Eukaryotes Requires Dpb11/TopBP1-GINS Interaction

Dpb11/Cut5/TopBP1 is evolutionarily conserved and is essential for the initiation of DNA replication in eukaryotes. The Dpb11 of the budding yeast Saccharomyces cerevisiae has four BRCT domains (BRCT1 to -4). The N-terminal pair (BRCT1 and -2) and the C-terminal pair (BRCT3 and -4) bind to cyclin-dependent kinase (CDK)-phosphorylated Sld3 and Sld2, respectively. These phosphorylation-dependent interactions trigger the initiation of DNA replication. BRCT1 and -2 and BRCT3 and -4 of Dpb11 are separated by a short stretch of ∼100 amino acids. It is unknown whether this inter-BRCT region functions in DNA replication. Here, we showed that the inter-BRCT region is a GINS interaction domain that is essential for cell growth and that mutations in this domain cause replication defects in budding yeast. We found the corresponding region in the vertebrate ortholog, TopBP1, and showed that the corresponding region also interacts with GINS and is required for efficient DNA replication. We propose that the inter-BRCT region of Dpb11 is a functionally conserved GINS interaction domain that is important for the initiation of DNA replication in eukaryotes.     

DNA Damage-Induced Accumulation of Centrosomal Chk1 Contributes to its Checkpoint Function

The checkpoint kinase Chk1 is an established transducer of ATR- and ATM-dependent signalling in response to DNA damage. In addition to its nuclear localization, Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B-Cdk1 during unperturbed cell cycles. Here, we demonstrate that DNA damage caused by ultraviolet irradiation or hydroxyurea treatment leads to centrosomal accumulation of endogenous Chk1 in normal human BJ fibroblasts and in ATR- or ATM-deficient fibroblasts. Chemical inhibition of ATR/ATM by caffeine led to enhanced centrosomal Chk1 deposition associated with nuclear Chk1 depletion. In contrast to normal or ATM-deficient fibroblasts, genetically ATR-deficient Seckel-fibroblasts showed detectable constitutive centrosomal accumulation of Chk1 even in the absence of exogenous insults. After DNA damage, the centrosomal fraction of Chk1 was found to be phosphorylated at ATR/ATM phosphorylation sites. Forced immobilization of kinase-inactive but not wild-type Chk1 to centrosomes resulted in a G2/M checkpoint defect. Finally, both DNA damage, and forced centrosomal expression of Chk1 in the absence of genotoxic treatments, induced centrosome amplification in a subset of cells, a phenomenon which could be suppressed by inhibition of ATM/ATR-mediated signaling. Taken together, our results suggest that accumulation of phosphorylated Chk1 at centrosomes constitutes an additional element in the DNA damage response. Centrosomal Chk1 induces G2/M cell cycle arrest and may evoke centrosome amplification, the latter possibly providing a backup mechanism for elimination of cells with impaired DNA damage checkpoints operating earlier during the cell cycle.     

Comprehensive analysis of cis- and trans-acting factors affecting ectopic Break-Induced Replication

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35–40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.     

端粒结合蛋白在端粒复制与损伤修复中的作用

端粒位于真核细胞线性染色体末端,正常的端粒长度与结构对于细胞基因组稳定的维持有重要作用.端粒DNA序列的高度重复性使其容易形成一些特殊的二级结构,相比染色体其他位置更难复制.结合在端粒上的Shelterin蛋白复合体由六个端粒结合蛋白组成,该复合体可以通过抑制端粒处异常DNA损伤修复途径的激活维持端粒的稳定.此外,近几...     

c-Myc Accelerates S-Phase and Requires WRN to Avoid Replication Stress

c-Myc interacts with components of the pre-replication complex and directly regulates DNA replication [1]. However the consequences of this novel c-Myc function on cell cycle dynamics and replication-associated damage are unknown. Here, we show that c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase while c-Myc deficient fibroblasts exhibit a prolonged S-phase. We also show that the Werner DNA helicase protein (WRN) plays a critical role in supporting c-Myc-driven S-phase, as depletion of WRN in c-Myc overexpressing cells increases DNA damage specifically at sites of DNA synthesis. This excess DNA damage activates a “replication stress” pathway involving ATR, CHK1, CHK2, and p53, leading to rapid senescence of WRN deficient c-Myc overexpressing cells. Indeed, depletion of p53 rescues this senescence response. We propose that WRN functions to repair abnormal replication structures caused by the acceleration of DNA replication by c-Myc. This work provides an additional mechanistic explanation for c-Myc-induced DNA damage and senescence, and reveals a vulnerability of c-Myc overexpressing cells that could potentially be exploited in cancer therapy.     

Efficient Replication of Epstein-Barr Virus-Derived Plasmids Requires Tethering by EBNA1 to Host Chromosomes

The EBNA1 protein of Epstein-Barr virus enables plasmids carrying oriP both to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element of oriP, DS (dyad symmetry); at another element, FR (family of repeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently from oriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supporting oriP functions. The implication is that EBNA1''s DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.