Interplay between Fanconi anemia and homologous recombination pathways in genome integrity

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error‐free pathway for double‐strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication‐associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.     

Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.     

Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

ING2 controls the progression of DNA replication forks to maintain genome stability

Inhibitor of growth 2 (ING2) is a candidate tumour suppressor gene the expression of which is frequently lost in tumours. Here, we identified a new function for ING2 in the control of DNA replication and in the maintenance of genome stability. Global replication rate was markedly reduced during normal S‐phase in small interfering RNA (siRNA) ING2 cells, as seen in a DNA fibre spreading experiment. Accordingly, we found that ING2 interacts with proliferating cell nuclear antigen and regulates its amount to the chromatin fraction, allowing normal replication progression and normal cell proliferation. Deregulation of DNA replication has been previously associated with genome instability. Hence, a high proportion of siRNA ING2 cells presented endoreduplication of their genome as well as an increased frequency of sister chromatid exchange. Thus, we propose for the first time that ING2 might function as a tumour suppressor gene by directly maintaining DNA integrity.     

ATR activation and replication fork restart are defective in FANCM‐deficient cells

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA‐ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR‐mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.     

Asymmetry of DNA replication fork progression in Werner's syndrome

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.     

Mta2 promotes Tipin-dependent maintenance of replication fork integrity

Orderly progression of S phase requires the action of replisome-associated Tipin and Tim1 proteins, whose molecular function is poorly understood. Here, we show that Tipin deficiency leads to the accumulation of aberrant replication intermediates known as reversed forks. We identified Mta2, a subunit of the NuRD chromatin remodeler complex, as a novel Tipin binding partner and mediator of its function. Mta2 is required for Tipin-dependent Polymerase α binding to replicating chromatin, and this function is essential to prevent the accumulation of reversed forks. Given the role of the Mta2–NuRD complex in the maintenance of heterochromatin, which is usually associated with hard-to-replicate DNA sequences, we tested the role of Tipin in the replication of such regions. Using a novel assay we developed to monitor replication of specific genomic loci in Xenopus laevis egg extract we demonstrated that Tipin is directly required for efficient replication of vertebrate centromeric DNA. Overall these results suggest that Mta2 and Tipin cooperate to maintain replication fork integrity, especially on regions that are intrinsically difficult to duplicate.     

SLX4: multitasking to maintain genome stability

The SLX4/FANCP tumor suppressor has emerged as a key player in the maintenance of genome stability, making pivotal contributions to the repair of interstrand cross-links, homologous recombination, and in response to replication stress genome-wide as well as at specific loci such as common fragile sites and telomeres. SLX4 does so in part by acting as a scaffold that controls and coordinates the XPF–ERCC1, MUS81–EME1, and SLX1 structure-specific endonucleases in different DNA repair and recombination mechanisms. It also interacts with other important DNA repair and cell cycle control factors including MSH2, PLK1, TRF2, and TOPBP1 as well as with ubiquitin and SUMO. This review aims at providing an up-to-date and comprehensive view on the key functions that SLX4 fulfills to maintain genome stability as well as to highlight and discuss areas of uncertainty and emerging concepts.     

RADX prevents genome instability by confining replication fork reversal to stalled forks

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Tolerating DNA damage during eukaryotic chromosome replication

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.     

Regulation of replication fork speed: Mechanisms and impact on genomic stability

Replication of DNA is a fundamental biological process that ensures precise duplication of the genome and thus safeguards inheritance. Any errors occurring during this process must be repaired before the cell divides, by activating the DNA damage response (DDR) machinery that detects and corrects the DNA lesions. Consistent with its significance, DNA replication is under stringent control, both spatial and temporal. Defined regions of the genome are replicated at specific times during S phase and the speed of replication fork progression is adjusted to fully replicate DNA in pace with the cell cycle. Insults that impair DNA replication cause replication stress (RS), which can lead to genomic instability and, potentially, to cell transformation. In this perspective, we review the current concept of replication stress, including the recent findings on the effects of accelerated fork speed and their impact on genomic (in)stability. We discuss in detail the Fork Speed Regulatory Network (FSRN), an integrated molecular machinery that regulates the velocity of DNA replication forks. Finally, we explore the potential for targeting FSRN components as an avenue to treat cancer.     

DCAF14 promotes stalled fork stability to maintain genome integrity

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The role of SMARCAL1 in replication fork stability and telomere maintenance

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.     

Histone deacetylases 1 and 2 regulate DNA replication and DNA repair: potential targets for genome stability-mechanism-based therapeutics for a subset of cancers

Histone deacetylases 1 and 2 (HDAC1,2) belong to the class I HDAC family, which are targeted by the FDA-approved small molecule HDAC inhibitors currently used in cancer therapy. HDAC1,2 are recruited to DNA break sites during DNA repair and to chromatin around forks during DNA replication. Cancer cells use DNA repair and DNA replication as survival mechanisms and to evade chemotherapy-induced cytotoxicity. Hence, it is vital to understand how HDAC1,2 function during the genome maintenance processes (DNA replication and DNA repair) in order to gain insights into the mode-of-action of HDAC inhibitors in cancer therapeutics. The first-in-class HDAC1,2-selective inhibitors and Hdac1,2 conditional knockout systems greatly facilitated dissecting the precise mechanisms by which HDAC1,2 control genome stability in normal and cancer cells. In this perspective, I summarize the findings on the mechanistic functions of class I HDACs, specifically, HDAC1,2 in genome maintenance, unanswered questions for future investigations and views on how this knowledge could be harnessed for better-targeted cancer therapeutics for a subset of cancers.     

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.     

FANCD2 influences replication fork processes and genome stability in response to clustered DSBs

Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.     

真核细胞DNA复制叉稳定性维持机制的研究进展

DNA复制是细胞最基本的生命活动之一,是生物体生存和繁殖的基础。从原核生物到真核生物,DNA复制过程基本保守,分为复制起始和延伸两个阶段。复制叉是DNA复制的基本结构,它容易遭受多种内源或外源的DNA复制压力影响而停顿,导致基因组不稳定,引起细胞凋亡、癌变或细胞死亡等严重后果。为了维持复制叉的稳定,细胞进化出了一系列机制,其中最重要机制之一便是S期细胞周期检验点。就影响DNA复制叉稳定的内外因素、S期细胞周期检验点与复制叉稳定性的关系以及复制叉稳定性与相关疾病的发生、治疗等问题进行简要综述。