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Xiaobin S. Wang Demis Menolfi Foon Wu-Baer Marco Fangazio Stefanie N. Meyer Zhengping Shao Yunyue Wang Yimeng Zhu Brian J. Lee Verna M. Estes Olivia M. Cupo Jean Gautier Laura Pasqualucci Riccardo Dalla-Favera Richard Baer Shan Zha 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(38)
CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4−/−Tp53−/− mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs−/−Tp53−/− mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.B cell lymphomas often carry oncogenic chromosomal translocations involving the immunoglobulin (Ig) genes, where programmed DNA double-strand breaks (DSBs) are created during the assembly and modifications of the Ig loci (1). The classical nonhomologous end-joining (cNHEJ) pathway of DSB repair is exclusively required for the assembly of functional Ig genes by V(D)J recombination. However, significant (up to 25 to 50%) class switch recombination (CSR) on the Ig heavy chain (IgH) can be achieved in cNHEJ-deficient cells via alternative end-joining (A-EJ), a distinct DSB repair pathway that preferentially uses microhomologies (MHs) at the junctions (2–6). In addition to CSR, the A-EJ pathway can also generate chromosomal translocations in reporter assays (7–9). DNA end resection that generates 3′ single-stranded DNA (ssDNA) overhangs (10) promotes A-EJ by exposing the flanking MHs. However, whether end resection is necessary for A-EJ–mediated oncogenic translocation and lymphomagenesis in vivo remains unknown.The C-terminal–binding protein (CtBP)-interacting protein (CtIP), like its yeast ortholog Sae2, initiates DNA end resection together with the MRE11-RAD50-NBS1 (MRN) nuclease complex (11). By virtue of its resection activity, CtIP was implicated in A-EJ (7–9). CtIP expression and protein levels are higher in S and G2 phases and lower in the G1 phase (12, 13). Like the MRN complex, CtIP is essential for murine development (14) and the proliferation of normal lymphocytes (4, 15, 16), rendering it difficult to examine its role during oncogenesis using null or conditional-null alleles. CtIP is phosphorylated by CDK at T847 in the S and G2 phases of the cell cycle (17) and by ATM and ATR kinases at T859 (T855 in mouse) and other sites upon DNA damage (18–21). While T847 phosphorylation of CtIP is essential for murine development (16), mice carrying an alanine substitution at the T855 phosphorylation site of CtIP (CtipT855A) develop normally with mild end resection defects (4, 15). Moreover, CtipT855A/T855A mice display normal lymphocyte development and proliferation (4, 15), providing a tool to test how CtIP and T855 phosphorylation contribute to chromosomal translocation and lymphomagenesis in vivo.The cNHEJ/Tp53 double-deficient mice routinely succumb to pro–B cell lymphomas bearing A-EJ–mediated IgH-Myc translocation and coamplification (22–24), providing an ideal model to examine the role of CtIP and end resection in A-EJ–mediated lymphomagenesis. Mechanistically, the initial translocation joins a RAG-initiated IgH DSB on chromosome 12 with sequences downstream of the c-Myc oncogene on chromosome 15 to form a dicentric (12, 15) chromosome (22). The dicentric intermediate breaks during mitosis, and the chromosome that contains the IgH-Myc translocation is joined with its sister to form a new dicentric chromosome, thereby initiating a breakage-fusion-bridge (BFB) cycle and eventually leading to a coamplification of IgH-Myc translocation under proliferation selection (1, 22). Since this occurs in cNHEJ-deficient cells, the initial translocation and the dicentric formation are both mediated by the A-EJ pathway. Tp53 deficiency is critical for the tolerance of the genomic instability and subsequent overexpression of the Myc oncogene. In addition to Xrcc4/Tp53-deficient mice, other cNHEJ/Tp53-deficient models, including Artemis- and DNA-PKcs–deficient mice, also develop pro–B cell lymphomas with IgH-Myc coamplification (24–27), although the exact organization of amplicons remains undetermined. In addition to these experimental pro–B cell lymphoma models, BFB cycles also underlie tumor initiation and drug resistance in other human cancers (28, 29).Here, we examine how CtIP-mediated end resection contributes to A-EJ–mediated chromosomal translocations and lymphomagenesis by characterizing cNHEJ/Tp53 double-deficient mice with or without the CtIP-T855A mutation. The results showed that the CtIP-T855A mutation causes neonatal lethality in Xrcc4−/−Tp53−/− mice without apparent lymphomas or hematopoietic failure. Instead, the CtIP-T855A mutation exacerbates the reduced mitotic index in Xrcc4−/−Tp53−/− olfactory neurons. In contrast, CtIPT855A/T855AKu70−/− mice are viable, although small even with wild-type (WT) Tp53 status. Moreover, the CtIP-T855A mutation delays lymphomagenesis and alters the tumor spectrum of DNA-PKcs−/−Tp53−/− mice. Yet, high-throughput genome-wide translocation sequencing (HTGTS) of RAG- and endonuclease-initiated DSBs shows that CtIP T855 phosphorylation is not required for hairpin opening or the initiation of end resection but consistently reduces the extent of end resection. Correspondingly, the CtIP-T855A mutation attenuates splenomegaly in the λ-Myc transgenic mouse model, suggesting a role for T855 phosphorylation in lymphomagenesis beyond translocation. In this context, CtIPT855A/T855A cells show no measurable defects in replication fork stability but consistent defects in IR-induced G2/M checkpoint maintenance. 相似文献
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Marchetti F Essers J Kanaar R Wyrobek AJ 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(45):17725-17729
Male and female germ cells can transmit genetic defects that lead to pregnancy loss, infant mortality, birth defects, and genetic diseases in offspring; however, the parental origins of transmitted defects are not random, with de novo mutations and chromosomal structural aberrations transmitted predominantly by sperm. We tested the hypotheses that paternal mutagenic exposure during late spermatogenesis can induce damage that persists in the fertilizing sperm and that the risk of embryos with paternally transmitted chromosomal aberrations depends on the efficiency of maternal DNA repair during the first cycle after fertilization. We show that female mice with defective DNA double-strand break repair had significantly increased frequencies of zygotes with sperm-derived chromosomal aberrations after matings with wild-type males irradiated 7 days earlier with 4 Gy of ionizing radiation. These findings demonstrate that mutagenic exposures during late spermatogenesis can induce damage that persists for at least 7 days in the fertilizing sperm and that maternal genotype plays a major role in determining the risks for pregnancy loss and frequencies of offspring with chromosomal defects of paternal origin. 相似文献
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Juan Huang Wenke Zhou Wei Dong Annie M. Watson Yang Hong 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(20):8284-8289
With the completion of genome sequences of major model organisms, increasingly sophisticated genetic tools are necessary for investigating the complex and coordinated functions of genes. Here we describe a genetic manipulation system termed “genomic engineering” in Drosophila. Genomic engineering is a 2-step process that combines the ends-out (replacement) gene targeting with phage integrase φC31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knock-out line is generated by deleting the target gene and replacing it with an integration site of φC31. Second, DNA integration by φC31 is used to reintroduce modified target-gene DNA into the native locus in the founder knock-out line. Genomic engineering permits directed and highly efficient modifications of a chosen genomic locus into virtually any desired mutant allele. We have successfully applied the genomic engineering scheme on 6 different genes and have generated at their loci more than 70 unique alleles. 相似文献
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Charlotte A. Cockram Milana Filatenkova Vincent Danos Meriem El Karoui David R. F. Leach 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(34):E4735-E4742
Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.DNA double-strand break repair (DSBR) is essential for cell survival and repair-deficient cells are highly sensitive to chromosome breakage. In Escherichia coli, a single unrepaired DNA DSB per replication cycle is lethal, illustrating the critical nature of the repair reaction (1). DSBR in E. coli is mediated by homologous recombination, which relies on the RecA protein to efficiently recognize DNA sequence identity between two molecules. RecA homologs are widely conserved from bacteriophages to mammals, where they are known as the Rad51 proteins (2). The RecA protein plays its central role by binding single-stranded DNA (ssDNA) to form a presynaptic filament that searches for a homologous double-stranded DNA (dsDNA) donor from which to repair. It then catalyzes a strand-exchange reaction to form a joint molecule (3), which is stabilized by the branch migration activities of the RecG and RuvAB proteins (4). The joint molecule is then resolved by cleavage at its four-way Holliday junction by the nuclease activity of RuvABC (5, 6).RecA binding at the site of a DSB is dependent on the activity of the RecBCD enzyme (Fig. 1A). RecBCD is a helicase-nuclease that binds to dsDNA ends, then separates and unwinds the two DNA strands using the helicase activities of the RecB and RecD subunits (see refs. 7 and 8 for recent reviews). RecD is the faster motor of the two and this consequently results in the formation of a ssDNA loop ahead of RecB (Loop 1 in Fig. 1A) (9). As the enzyme translocates along dsDNA, the 3′-terminated strand is continually passed through the Chi-scanning site thought to be located in the RecC protein (10). When a Chi sequence (the octamer 5′-GCTGGTGG-3′) enters this recognition domain, the RecD motor is disengaged and the 3′ strand continues to be unwound by RecB. Under in vitro conditions, where the concentration of magnesium exceeds that of ATP, the 3′ end (unwound by RecB) is rapidly digested before Chi recognition, whereas the 5′ end (unwound by RecD) is intermittently cleaved (11, 12). After Chi recognition the 3′ end is no longer cleaved but the nuclease domain of RecB continues to degrade the 5′ end as it exits the enzyme (11, 12). Under in vitro conditions where the concentration of ATP exceeds that of magnesium, unwinding takes place but the only site of cleavage detected is ∼5 nucleotides 3′ of the Chi sequence (13, 14). Because the RecB motor continues to operate while the RecD motor is disengaged, Loop 1 is converted to a second loop located between the RecB and RecC subunits or to a tail upon release of the Chi sequence from its recognition site. We therefore describe this single-stranded region as Loop/Tail 2 in Fig. 1A. After the whole of Loop 1 is converted to Loop/Tail 2, this second single-stranded region continues to grow as long as the RecB subunit unwinds the dsDNA. The RecBCD enzyme enables RecA protein to load on to Loop/Tail 2 to generate the presynaptic filament necessary to search for homology and initiate strand-exchange (15). Finally, the RecBCD enzyme stops translocation and disassembles as it dissociates from the DNA, releasing a DNA-free RecC subunit (16).Open in a separate windowFig. 1.DSBR in E. coli. (A and B) Schematic representation of DSB processing by the RecBCD complex. (A) Before Chi recognition, both the RecB and RecD motors progress along the DNA. RecD is the faster motor and as a result a loop of ssDNA (Loop 1) is formed ahead of the slower RecB motor. The 3′ ssDNA strand is scanned for the Chi sequence by the RecC protein. (B) After Chi recognition, RecBCD likely undergoes a conformational change so that only the RecB motor is engaged. The RecA protein is recruited by the RecB nuclease domain and loaded onto the ssDNA loop generated by RecB unwinding to promote RecA nucleoprotein filament formation. In this schematic representation, the Chi site is shown held in its recognition site. However, the Chi site will be released either by disassembly of the RecBCD complex or at some point before this and the second single-stranded region on the 3′ terminating strand will be converted from a loop to a tail. Therefore, this region is denoted Loop/Tail 2. The mathematical model described in SI Appendix does not depend on the ATP/magnesium dependent differential cleavage of DNA strands (7, 8), nor does it depend on the precise time that the 3′ end is released from the complex following Chi recognition. (C) The hairpin endonuclease SbcCD is used to cleave a 246-bp interrupted palindrome inserted in the lacZ gene of the E. coli chromosome. Cleavage of this DNA hairpin results in the generation of a site-specific DSB on only one of a pair of replicating sister chromosomes, thus leaving an intact sister chromosome to serve as a template for repair by homologous recombination.Our understanding of the action of RecBCD and RecA has been the result of more than 40 years of genetic analysis and biochemical investigation of these purified proteins in vitro. However, relatively little is known about their activities on the genomic scale. To investigate these reactions in vivo, we have used RecA chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) in an experimental system that allows us to introduce a single and fully repairable DSB into the chromosome of E. coli (1). Because DSBR by homologous recombination normally involves the repair of a broken chromosome by copying the information on an unbroken sister chromosome, our laboratory has previously developed a procedure for the cleavage of only one copy of two genetically identical sister chromosomes (1). We have made use of the observation that the hairpin nuclease SbcCD specifically cleaves only one of the two sister chromosomes following DNA replication through a 246-bp interrupted palindrome to generate a two-ended DSB (1). As shown in Fig. 1B, this break is fully repairable and we have shown that recombination-proficient cells suffer very little loss of fitness in repairing such breaks (17).Here we investigate in vivo and in a quantitative manner the first steps of DSBR: because the outcome of RecBCD action is understood to be the loading of RecA on DNA in a Chi-dependent manner, we use RecA-ChIP to reveal the consequences of RecBCD action on a genomic scale during DSBR. Analyses of most ChIP-Seq datasets focus on the identification of regions of significant enrichment of a given protein but do not take into account the underlying mechanisms giving rise to the binding (18). We reasoned that given the detailed mechanistic understanding of RecBCD in vitro, we could gain a deeper insight into its in vivo functions by developing a mathematical model of RecBCD action that would enable us to estimate the mechanistic parameters of the complex in live cells. Our ChIP data indicate that RecA is indeed loaded on to DNA in a Chi-dependent manner and we have used our mathematical model to infer the parameters of RecBCD action in vivo on a genomic scale. Furthermore, our analysis reveals that DSBR at lacZ induces DSBR in the terminus region of the chromosome, an unanticipated observation illuminated by the genomic scale of our data. 相似文献
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Vespa L Warrington RT Mokros P Siroky J Shippen DE 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(46):18145-18150
Telomeres have the paradoxical ability of protecting linear chromosome ends from DNA damage sensors by using these same proteins as essential components of their maintenance machinery. We have previously shown that the absence of ataxia telangiectasia mutated (ATM), a central regulator of the DNA damage response, accelerates the onset of genome instability in telomerase-deficient Arabidopsis, without increasing the rate of bulk telomere shortening. Here, we examine individual telomere tracts through successive plant generations using both fluorescence situ in hybridization (FISH) and primer extension telomere repeat amplification (PETRA). Unexpectedly, we found that the onset of profound developmental defects and abundant end-to-end chromosome fusions in fifth generation (G(5)) atm tert mutants required the presence of only one critically shortened telomere. Parent progeny analysis revealed that the short telomere arose as a consequence of an unusually large telomere rapid deletion (TRD) event. The most dramatic TRD was detected in atm tert mutants that had undergone meiosis. Notably, in contrast to TRD, alternative lengthening of telomeres (ALT) was suppressed in the absence of ATM. Finally, we show that size differences between telomeres on homologous chromosome ends are greater for atm tert than tert plants. Altogether, these findings suggest a dual role for ATM in regulating telomere size by promoting elongation of short telomeres and by preventing the accumulation of cells that harbor large telomere deletions. 相似文献
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Zhang J McCabe KA Bell CE 《Proceedings of the National Academy of Sciences of the United States of America》2011,108(29):11872-11877
The λ exonuclease is an ATP-independent enzyme that binds to dsDNA ends and processively digests the 5′-ended strand to form 5′ mononucleotides and a long 3′ overhang. The crystal structure of λ exonuclease revealed a toroidal homotrimer with a central funnel-shaped channel for tracking along the DNA, and a mechanism for processivity based on topological linkage of the trimer to the DNA was proposed. Here, we have determined the crystal structure of λ exonuclease in complex with DNA at 1.88-Å resolution. The structure reveals that the enzyme unwinds the DNA prior to cleavage, such that two nucleotides of the 5′-ended strand insert into the active site of one subunit of the trimer, while the 3′-ended strand passes through the central channel to emerge out the back of the trimer. Unwinding of the DNA is facilitated by several apolar residues, including Leu78, that wedge into the base pairs at the single/double-strand junction to form favorable hydrophobic interactions. The terminal 5′ phosphate of the DNA binds to a positively charged pocket buried at the end of the active site, while the scissile phosphate bridges two active site Mg2+ ions. Our data suggest a mechanism for processivity in which wedging of Leu78 and other apolar residues into the base pairs of the DNA restricts backward movement, whereas attraction of the 5′ phosphate to the positively charged pocket drives forward movement of the enzyme along the DNA at each cycle of the reaction. Thus, processivity of λ exonuclease operates not only at the level of the trimer, but also at the level of the monomer. 相似文献
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目的 :观察原位细胞凋亡与急性白血病 (AL )发生与转归的关系。方法 :用碱性磷酸酶抗碱性磷酸酶(APAAP)免疫酶标和 DNA末端原位标记 (ISEL )双重染色方法检测 49例急性白血病骨髓涂片中细胞凋亡状况 ,初治组 2 3例与 15例缺铁性贫血病例对照并与 2 6例经治病例对照。结果 :未经治疗的 AL 平均凋亡指数 (AI)明显低于对照组 (P <0 .0 1)。化疗后凋亡明显增加 ,与化疗前比较差异有极显著性意义 (P <0 .0 1)。AI>5 %者平均原始细胞下降指数 (MBDI)为 2 8.6 8% ,AI<5 %者平均 MBDI仅为 4.9%。凋亡细胞大多数为白血病细胞。非白血病细胞凋亡亦有增加。结论 :AL 发生与凋亡逃逸有关 ,细胞毒化疗的主要机理之一为促进细胞凋亡。化疗后全血细胞下降与正常造血细胞凋亡增加有关。 相似文献
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The ability to stably introduce genes into the germline of animals provides a powerful means to address the genetic basis
of physiology. Introduction of genes to generate transgenic animals has facilitated the development of complex genetic models
of disease, as well as the in vivo study of gene function. However, one drawback of traditional transgenic technologies in
which genes are microinjected into early-stage embryos is that there is little control over where and in how many copies genes
are introduced into the genome. The development of animal transgenic technologies, which take advantage of homologous recombination
mechanisms and the manipulation of embryonic stem (ES) cells, allows investigators to target and alter specific loci. In mouse
transgenic systems, a plethora of sophisticated gene-targeting strategies now permit investigators to manipulate the genome
in ways that essentially allow one to introduce virtually any desired change into the genome. Fur-thermore, when coupled with
systems that allow for conditional gene expression, these gene-targeting strategies allow both temporal and tissue specific
control of alterations to the genome. In the present review we briefly discuss some of the more recent gene-targeting strategies
that have been developed to address the limitations of traditional animal transgenesis. 相似文献
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The DNA damage response (DDR) maintains genomic integrity through an elaborate network of signaling pathways that sense DNA damage and recruit effector factors to repair damaged DNA. DDR signaling pathways are usurped and manipulated by the replication programs of many viruses. Here, we review the papillomavirus (PV) life cycle, highlighting current knowledge of how PVs recruit and engage the DDR to facilitate productive infection. 相似文献
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Xuejie Wang Yang Dong Xiaocong Zhao Jinbao Li Jordan Lee Zhenxin Yan Shuangshuang Yang Wenqiang Wu Ximiao Hou Guangxue Liu Yueyue Zhang Lun Song Gang Cai Qing Li Grzegorz Ira Xinghua Zhang Xuefeng Chen 《Proceedings of the National Academy of Sciences of the United States of America》2021,118(25)
Single-stranded DNA (ssDNA) covered with the heterotrimeric Replication Protein A (RPA) complex is a central intermediate of DNA replication and repair. How RPA is regulated to ensure the fidelity of DNA replication and repair remains poorly understood. Yeast Rtt105 is an RPA-interacting protein required for RPA nuclear import and efficient ssDNA binding. Here, we describe an important role of Rtt105 in high-fidelity DNA replication and recombination and demonstrate that these functions of Rtt105 primarily depend on its regulation of RPA. The deletion of RTT105 causes elevated spontaneous DNA mutations with large duplications or deletions mediated by microhomologies. Rtt105 is recruited to DNA double-stranded break (DSB) ends where it promotes RPA assembly and homologous recombination repair by gene conversion or break-induced replication. In contrast, Rtt105 attenuates DSB repair by the mutagenic single-strand annealing or alternative end joining pathway. Thus, Rtt105-mediated regulation of RPA promotes high-fidelity replication and recombination while suppressing repair by deleterious pathways. Finally, we show that the human RPA-interacting protein hRIP-α, a putative functional homolog of Rtt105, also stimulates RPA assembly on ssDNA, suggesting the conservation of an Rtt105-mediated mechanism.Faithful DNA replication and repair are essential for the maintenance of genetic material (1). Even minor defects in replication or repair can cause high loads of mutations, genome instability, cancer, and other diseases (1). Deficiency in different DNA repair or replication proteins can lead to distinct mutation patterns (2–4). For example, deficiency in mismatch repair results in increased microsatellite instability, while deficiency in homologous recombination repair is often associated with tandem duplications or deletions (3–7). Sequence analysis of various cancer types has identified many distinct genome rearrangement and mutation signatures (8). However, the genetic basis for some of these signatures remains poorly understood, thus requiring further investigation in experimental models (8).In eukaryotic cells, Replication Protein A (RPA), the major single-stranded DNA (ssDNA) binding protein complex, is essential for DNA replication, repair, and recombination (9–13). It is also crucial for the suppression of mutations and genome instability (14–17). RPA acts as a key scaffold to recruit and coordinate proteins involved in different DNA metabolic processes (14, 15, 17). As the first responder of ssDNA, RPA participates in both replication initiation and elongation (10, 12, 13). During replication or under replication stresses, the exposed ssDNA must be protected and stabilized by RPA to prevent formation of secondary structures (14, 16). RPA is also essential for DNA double-stranded break (DSB) repair by the homologous recombination (HR) pathway (18–21). During HR, the 5′-terminated strands of DSBs are initially processed by the resection machinery, generating 3′-tailed ssDNA (22). The 3′-ssDNA becomes bound by the RPA complex to activate the DNA damage checkpoint (23). RPA is subsequently replaced by the Rad51 recombinase to form a Rad51 nucleoprotein filament (19, 24). This recombinase filament catalyzes invasion of the 3′-strands at the homologous sequence to form the D-loop structure, followed by repair DNA synthesis and resolution of recombination intermediates (18, 19, 24). During HR, RPA prevents the formation of DNA secondary structures and protects 3′-ssDNA from nucleolytic degradation (25). In addition, recent work implies a role of RPA in homology recognition (26).RPA is composed of three subunits, Rfa1, Rfa2, and Rfa3, and with a total of six oligonucleotide-binding (OB) motifs that mediate interactions with ssDNA or proteins (14, 17, 27). RPA can associate with ssDNA in different modes (28). It binds short DNA (8 to 10 nt) in an unstable mode and longer ssDNA (28 to 30 nt) in a high-affinity mode (28–31). Recent single-molecule studies revealed that RPA binding on ssDNA is highly dynamic (28, 32). It can rapidly diffuse within the bound DNA ligand and quickly exchange between the free and ssDNA-bound states (32–35). The cellular functions of RPA rely on its high ssDNA-binding affinity and its ability to interact with different proteins (28). Although RPA has a high affinity for ssDNA, recent studies have suggested that the binding of RPA on chromatin requires additional regulations (36). How RPA is regulated to ensure replication and repair fidelity remains poorly understood.Rtt105, a protein initially identified as a regulator of the Ty1 retrotransposon, has recently been shown to interact with RPA and acts as an RPA chaperone (36). It facilitates the nuclear localization of RPA and stimulates the loading of RPA at replication forks in unperturbed conditions or under replication stresses (36). Rtt105 exhibits synthetic genetic interactions with genes encoding replisome proteins and is required for heterochromatin silencing and telomere maintenance (37). The deletion of RTT105 results in increased gross chromosomal rearrangements and reduced resistance to DNA-damaging agents (36, 38). In vitro, Rtt105 can directly stimulate RPA binding to ssDNA, likely by changing the binding mode of RPA (36).In this study, by using a combination of genetic, biochemical, and single-molecule approaches, we demonstrate that Rtt105-dependent regulation of RPA promotes high-fidelity genome duplication and recombination while suppressing mutations and the low-fidelity repair pathways. We provide evidence that human hRIP-α, the putative functional homolog of yeast Rtt105, could regulate human RPA assembly on ssDNA in vitro. Our study unveils a layer of regulation on the maintenance of genome integrity that relies on dynamic RPA binding on ssDNA to ensure high-fidelity replication or recombination. 相似文献
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Differential expression of homologous recombination DNA repair genes in the early and advanced stages of myelodysplastic syndrome
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Jan Valka Jitka Vesela Hana Votavova Michaela Dostalova‐Merkerova Zuzana Horakova Vit Campr Jana Brezinova Zuzana Zemanova Anna Jonasova Jaroslav Cermak Monika Belickova 《European journal of haematology》2017,99(4):323-331
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Logan R. Myler Ignacio F. Gallardo Yi Zhou Fade Gong Soo-Hyun Yang Marc S. Wold Kyle M. Miller Tanya T. Paull Ilya J. Finkelstein 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(9):E1170-E1179
Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.All DNA maintenance processes require nucleases, which enzymatically cleave the phosphodiester bonds in nucleic acids. Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1–3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2, 4–7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8, 9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1, 10–12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis. The central role of Exo1 in DNA repair is highlighted by the large set of genetic interactions between Exo1 and nearly all other DNA maintenance and metabolism pathways (13).Exo1 generates long tracts of ssDNA in both MMR and DSB repair (3). This ssDNA is rapidly bound by replication protein A (RPA), a ubiquitous heterotrimeric protein that participates in all DNA transactions that generate ssDNA intermediates (14). RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15–17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/DNA2- and Exo1-dependent DNA resection pathways (18–21). Reconstitution of both the yeast and human BLM (Sgs1 in yeast)/DNA2-dependent resection reactions established that RPA stimulates DNA unwinding by BLM/Sgs1 and enforces a 5′-endonuclease polarity on DNA2 (20, 22). However, the effect of RPA on Exo1 remains unresolved. Independent studies using reconstituted yeast proteins reported that RPA could both inhibit (23) and stimulate yeast Exo1 (yExo1) (18). Similarly, human RPA has variously been reported to stimulate (19) or inhibit human Exo1 (hExo1) (4, 5, 21).In addition to RPA, human cells also encode SOSS1, a heterotrimeric ssDNA-binding complex that is essential for HR (24). SOSS1 consists of INTS3 (SOSSA), hSSB1 (SOSSB1), and C9orf80 (SOSSC) (24–26). SOSSB1 encodes a single ssDNA-binding domain that bears structural homology to Escherichia coli ssDNA-binding protein (SSB) (24). SOSS1 foci form rapidly after induction of DNA breaks, and ablation of SOSS1 severely reduces DNA resection, γH2AX foci formation, and HR at both ionizing radiation- and restriction endonuclease-induced DSBs (12, 24, 25, 27). In vitro, SOSS1 stimulates hExo1-mediated DNA resection and may help to load hExo1 at ss/dsDNA junctions (21). However, the functional relationship between SOSS1 and RPA during hExo1 resection remains unresolved.Here, we use high-throughput single-molecule DNA curtains and quantitative cell biology to reveal the interplay between human and yeast Exo1 and SSBs during DNA resection. We show that both human and yeast Exo1s are processive nucleases, but are rapidly stripped from DNA by RPA. RPA inhibition is dependent on its multiple DNA binding domains. Remarkably, SOSS1 and other SSBs with fewer than three DNA binding domains support long-range resection by hExo1. In human cells, depletion of RPA increases the rate of hExo1 recruitment to laser-induced DNA damage but reduces the extent of resection. In the presence of RPA, both human and yeast Exo1 can resect DNA using a distributive, multiple-turnover mechanism, potentially reconciling prior conflicting in vitro observations. Together, our work reveals the mechanistic basis for how RPA and SOSS1 differentially modulate hExo1 activity and highlights an additional, unexpected role for these SSBs in DNA resection. We anticipate that these findings will shed light on how Exo1 is regulated in multiple genome maintenance pathways. 相似文献
18.
Deriano L Merle-Béral H Guipaud O Sabatier L Delic J 《British journal of haematology》2006,133(5):520-525
Non-homologous end joining (NHEJ) is an important determinant of genomic stability in mammalian cells. This DNA repair pathway is upregulated in a subset of B-cell chronic lymphocytic leukaemia (B-CLL) cells resistant to DNA damage-induced apoptosis. Using an in vitro assay for double-strand breaks (DSB) end ligation, we studied the fidelity of DSB repair in B-CLL cells which were resistant or sensitive to in vitro DSB-induced apoptosis with concomitant patients' resistance or sensitivity to chemotherapy, respectively. The fidelity of DNA repair was determined by DNA sequencing of polymerase chain reaction products cloned in pGEM-T vector. Sequence analysis of DNA end junctions showed that the frequency of accurate ligation was higher in sensitive B-CLL cells and control cell lines, than in resistant cells where end joining was associated with extended deletions. Upregulated and error-prone NHEJ in resistant cells could be a quite possible mechanism underlying both genomic instability and poor clinical outcome. 相似文献
19.
Bai H Sun M Ghosh P Hatfull GF Grindley ND Marko JF 《Proceedings of the National Academy of Sciences of the United States of America》2011,108(18):7419-7424
Structural and topological data suggest that serine site-specific DNA recombinases exchange duplex DNAs by rigid-body relative rotation of the two halves of the synapse, mediated by a flat protein-protein interaction surface. We present evidence for this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, from a single-DNA-based supercoil-release assay that allows us to follow crossover site cleavage, rotation, religation, and product release in real time. We have also used a two-DNA braiding-relaxation experiment to observe the effect of synapse rotation in reactions on two long molecules. Relaxation and unbraiding are rapid (averaging 54 and 70 turns/s, respectively) and complete, with no discernible pauses. Nevertheless, the molecular friction associated with rotation is larger than that of type-I topoisomerases in a similar assay. Surprisingly we find that the synapse can stay rotationally "open" for many minutes. 相似文献
20.
Elena Prisciandaro Laurens J. Ceulemans Dirk E. Van Raemdonck Herbert Decaluw Paul De Leyn Luca Bertolaccini 《Journal of thoracic disease》2022,14(7):2677
BackgroundPulmonary metastasectomy (PM) with curative intent has become a widely accepted treatment for lung metastases from solid tumours in selected patients, with low perioperative morbidity and mortality. In particular, PM is strongly recommended in selected patients with secondary lesions from colorectal cancer (CRC), due to its excellent postoperative prognosis. Nevertheless, the impact of the extent of PM on recurrence and survival remains controversial. This review aimed at assessing differences in short- and long-term postoperative outcomes depending on the extent of lung resection for lung metastases.MethodsA systematic literature review of studies comparing anatomical and non-anatomical resections of lung metastases was performed (Prospective Register of Systematic Reviews Registration: 254931). A literature search for articles published in English between the date of database inception and January 31, 2021 was performed in EMBASE (via Ovid), MEDLINE (via PubMed) and Cochrane CENTRAL. Retrospective studies, randomised and non-randomised controlled trials were included. The Cochrane Collaboration tool was used to determine the risk of bias for the primary outcome for included studies.ResultsOut of 432 papers, three retrospective non-randomised studies (1,342 patients) were selected for systematic reviewing. Although our search design did not exclude any primary tumour histology, all selected studies investigated surgical resection of lung metastases from CRC. Because of variations in the compared surgical approaches to pulmonary metastases, a meta-analysis proved unfeasible. There was a tendency to perform anatomical resections for larger metastases. Multivariate analyses revealed that anatomical resections were protective for recurrence-free survival (RFS), while the impact of such procedures on overall survival (OS) remained uncertain. A significantly higher incidence of resection-margin recurrences was observed in patients who underwent non-anatomical resections.DiscussionAnatomical resections of lung metastases from CRC seem to be associated with improved RFS. However, well-constructed comparative clinical trials focusing on the extent of PM are needed. 相似文献