AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。     

植物DNA双链断裂修复的保守性和特异性

文章概述了植物DNA双链断裂（double-strand break，DSB）修复的研究进展。从酵母、脊椎动物、植物在此领域已取得的成果来看，真核生物DSB修复在过程和参与蛋白方面均有一定的进化保守性；另一方面，植物的DSB修复有其特异之处。     

Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.     

Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells

Mammalian cells are able to repair chromosomal double-strand breaks (DSBs) both by homologous recombination and by mechanisms that require little or no homology. Although spontaneous homologous recombination is rare, DSBs will stimulate recombination by 2 to 3 orders of magnitude when homology is provided either from exogenous DNA in gene-targeting experiments or from a repeated chromosomal sequence. Using a gene-targeting assay in mouse embryonic stem cells, we now investigate the effect of heterology on recombinational repair of DSBs. Cells were cotransfected with an endonuclease expression plasmid to induce chromosomal DSBs and with substrates containing up to 1.2% heterology from which to repair the DSBs. We find that heterology decreases the efficiency of recombinational repair, with 1.2% sequence divergence resulting in an approximately sixfold reduction in recombination. Gene conversion tract lengths were examined in 80 recombinants. Relatively short gene conversion tracts were observed, with 80% of the recombinants having tracts of 58 bp or less. These results suggest that chromosome ends in mammalian cells are generally protected from extensive degradation prior to recombination. Gene conversion tracts that were long (up to 511 bp) were continuous, i.e., they contained an uninterrupted incorporation of the silent mutations. This continuity suggests that these long tracts arose from extensive degradation of the ends or from formation of heteroduplex DNA which is corrected with a strong bias in the direction of the unbroken strand.     

Two Forms of Repair of DNA in Mammalian Cells following Irradiation

When Chinese hamster cells are lysed on top of an alkaline sucrose gradient, in time a fairly discrete DNA-containing molecular species is released from an apparently more complex material. Small doses of X-radiation speed the resolution of this complex while large doses degrade the material released from it. Incubation after irradiation reverses both effects.     

Evidence for Conservative (Two-Progeny) DNA Double-Strand Break Repair

The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage λ Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.     

Pathways for Double-strand Break Repair in Genetically Unstable Z-DNA-forming Sequences

DNA can adopt many structures that differ from the canonical B-form, and several of these non-canonical DNA structures have been implicated in genetic instability associated with human disease. Earlier, we found that Z-DNA causes DNA double-strand breaks (DSBs) in mammalian cells that can result in large-scale deletions and rearrangements. In contrast, the same Z-DNA-forming CG repeat in Escherichia coli resulted in only small contractions or expansions within the repeat. This difference in the Z-DNA-induced mutation spectrum between mammals and bacteria might be due to different mechanisms for DSB repair; in mammalian cells, non-homologous end-joining (NHEJ) is a major DSB repair pathway, while E. coli do not contain this system and typically use homologous recombination (HR) to process DSBs. To test the extent to which the different DSB repair pathways influenced the Z-DNA-induced mutagenesis, we engineered bacterial E.coli strains to express an inducible NHEJ system, to mimic the situation in mammalian cells. Mycobacterium tuberculosis NHEJ proteins Ku and ligase D (LigD) were expressed in E.coli cells in the presence or absence of HR, and the Z-DNA-induced mutations were characterized. We found that the presence of the NHEJ mechanism markedly shifted the mutation spectrum from small deletions/insertions to large-scale deletions (from 2% to 24%). Our results demonstrate that NHEJ plays a role in the generation of Z-DNA-induced large-scale deletions, suggesting that this pathway is associated with DNA structure-induced destabilization of genomes from prokaryotes to eukaryotes.     

N Terminus of CtIP Is Critical for Homologous Recombination-mediated Double-strand Break Repair

DNA double-strand breaks (DSBs) represent one of the most lethal types of DNA damage cells encounter. CtIP (also known as RBBP8) acts together with the MRN (MRE11-RAD50-NBS1) complex to promote DNA end resection and the generation of single-stranded DNA, which is critically important for homologous recombination repair. However, it is not yet clear exactly how CtIP participates in this process. Here, we demonstrate that besides the known conserved C terminus, the N terminus of CtIP protein is also required in DSB end resection and DNA damage-induced G₂/M checkpoint control. We further show that both termini of CtIP can interact with the MRN complex and that the N terminus of CtIP, especially residues 22–45, binds to MRN and plays a critical role in targeting CtIP to sites of DNA breaks. Collectively, our results highlight the importance of the N terminus of CtIP in directing its localization and function in DSB repair.     

HDF1 and RAD17 Genes are Involved in DNA Double-strand Break Repair in Stationary Phase Saccharomyces cerevisiae

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex—the Rad17/Mec3/Ddc1 clamp—acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17Δ/rad17Δ, and hdf1Δ Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30°C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with ⁶⁰Co γ rays at 5 Gy/s, 50 Gy ≤ Dabs ≤ 200 Gy. Thereafter, cells were incubated in PBS (liquid holding: LH, 0 ≤ t ≤ 24 h). DNA chromosomal analysis (by pulsed-field electrophoresis), and surviving fractions were determined as a function of absorbed doses, either immediately after irradiation or after LH. Our results demonstrated that the proteins Rad17, as well as Hdf1, play essential roles in DBS repair and survival after gamma irradiation in the late stationary phase and upon nutrient stress (LH after irradiation). In haploid cells, the main pathway is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17. Results are compatible with the action of a network of DBS repair pathways expressed upon different ploidies, and different magnitudes of DNA damage.     

Kinetics of DNA Repair in Ultraviolet-Irradiated and N-acetoxy-2-acetylaminofluorene-Treated Mammalian Cells

Repair kinetics after saturating doses of ultraviolet radiation (UV), N-acetoxy-2-acetylaminofluorene (AAAF), and combinations of both agents were studied in human fibroblasts proficient and deficient in excision repair, and in Chinese hamster cells (V-79) deficient in excision repair. Three techniques were used: unscheduled DNA synthesis, photolysis of DNA repaired in the presence of bromodeoxyuridine (BrdUrd), and measurements of sites sensitive to a UV-endonuclease. The repair rate appears to be approximately constant during the first few hours after treatment. Later there is a decrease with time; the magnitude of the decrease depends on the cell line. Our data show that the decrease in repair observed in repair-deficient cells treated with combinations of both agents as compared to separate treatments is due neither to the cytotoxic effects of the agents used, nor to a shutoff of the repair system by the high concentrations of AAAF employed.     

Replicating Units (Replicons) of DNA in Cultured Mammalian Cells

Exponentially growing L5178Y mouse leukemic cells were incubated in the presence of 5′-bromodeoxyuridine (BUdR) for about 4 hr, transferred to the nonBUdR-containing medium for a certain period (t hours), and then pulse-labeled with TdR-³H for 10 min. When DNA isolated from these cells was subjected to CsCl gradient centrifugation, the ³H-activity was found to shift gradually from the heavy BUdR-containing peak to the light nonBUdR-containing peak with increasing time t. The average time required for the complete shift of ³H-activity from the heavy to the light DNA fraction was 2.76 hr. Taking this as the average replicating time and the size of DNA fragments in the present preparation as 1.3 × 10⁷ daltons, the rate of replication was found to be 2.1 nucleotides per strand per replicon per sec. By taking the upper limit of the average replicating time as the S period (7.3 hr), various characteristics of the replicating units, such as the lower and upper limits of average size, the average replicating time, the average number of replicating units, etc., were calculated (see Table I).     

The RING Finger Protein RNF8 Ubiquitinates Nbs1 to Promote DNA Double-strand Break Repair by Homologous Recombination

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.     

BRCA1与DSB修复路径取向

乳腺癌易感基因1(BRCA1)是一个肿瘤抑制基因.BRCA1参与DNA末端切除、细胞周期调控以及染色体修饰等来维护基因组的稳定性.有研究表明,它能够促进正确的DNA双链断裂(DSBs)修复,如同源重组修复(HDR)和经典的非同源末端连接(C-NHEJ);而抑制错误性的DSB修复,如单链退火修复(SSA)和非经典的末端连接(A-EJ);其机制是通过与某些DNA修复相关蛋白质的相互作用来引导DSB修复.目前,BRCA1在DSB修复通路中的作用机制尚未完全明确,仍有待进一步的研究.本文主要阐述BRCA1在DSB各修复通路中是如何发挥其引导作用的.     

组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用

DNA双链断裂(double strand breaks, DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶（histone deacetylase）作为抗肿瘤治疗的新靶标,其抑制剂(histonedeacetylase inhibitors, HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.     

53BP1与DNA双链断裂损伤修复

DNA的精确复制和遗传对维持基因组稳定性有重要作用。DNA双链断裂损伤可能诱导细胞凋亡和染色质重排,在肿瘤的发生发展过程中发挥作用。53BP1是DNA双链断裂修复中的重要调节蛋白质之一,对调控损伤修复平衡和维持基因组稳定性起着重要作用。本文主要对53BP1的结构、生物学功能、信号通路、分子机制和翻译后修饰做一浅显的总结和展望,希望能为53BP1的深入研究提供一些理论基础。