Mechanism of RAD51-dependent DNA interstrand cross-link repair

DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.     

The fission yeast FANCM ortholog directs non-crossover recombination during meiosis

The formation of healthy gametes depends on programmed DNA double-strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favor of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to humans suggests that this interplay may be a general feature of meiotic recombination.     

Robust crossover assurance and regulated interhomolog access maintain meiotic crossover number

Most organisms rely on interhomolog crossovers (COs) to ensure proper meiotic chromosome segregation but make few COs per chromosome pair. By monitoring repair events at a defined double-strand break (DSB) site during Caenorhabditis elegans meiosis, we reveal mechanisms that ensure formation of the obligate CO while limiting CO number. We find that CO is the preferred DSB repair outcome in the absence of inhibitory effects of other (nascent) recombination events. Thus, a single DSB per chromosome pair is largely sufficient to ensure CO formation. Further, we show that access to the homolog as a repair template is regulated, shutting down simultaneously for both CO and noncrossover (NCO) pathways. We propose that regulation of interhomolog access limits CO number and contributes to CO interference.     

Postreplicative formation of cohesion is required for repair and induced by a single DNA break

Sister-chromatid cohesion, established during replication by the protein complex cohesin, is essential for both chromosome segregation and double-strand break (DSB) repair. Normally, cohesion formation is strictly limited to the S phase of the cell cycle, but DSBs can trigger cohesion also after DNA replication has been completed. The function of this damage-induced cohesion remains unknown. In this investigation, we show that damage-induced cohesion is essential for repair in postreplicative cells in yeast. Furthermore, it is established genome-wide after induction of a single DSB, and it is controlled by the DNA damage response and cohesin-regulating factors. We thus define a cohesion establishment pathway that is independent of DNA duplication and acts together with cohesion formed during replication in sister chromatid-based DSB repair.     

Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing

Bloom syndrome, characterized by a predisposition to cancer, is caused by mutation of the RecQ DNA helicase gene BLM. The precise function of BLM remains unclear. Previous research suggested that Drosophila BLM functions in the repair of DNA double-strand breaks. Most double-strand breaks in flies are repaired by homologous recombination through the synthesis-dependent strand-annealing pathway. Here, we demonstrate that Drosophila BLM mutants are severely impaired in their ability to carry out repair DNA synthesis during synthesis-dependent strand annealing. Consequently, repair in the mutants is completed by error-prone pathways that create large deletions. These results suggest a model in which BLM maintains genomic stability by promoting efficient repair DNA synthesis and thereby prevents double-strand break repair by less precise pathways.     

DNA gyrase and the supercoiling of DNA

Negative supercoiling of bacterial DNA by DNA gyrase influences all metabolic processes involving DNA and is essential for replication. Gyrase supercoils DNA by a mechanism called sign inversion, whereby a positive supercoil is directly inverted to a negative one by passing a DNA segment through a transient double-strand break. Reversal of this scheme relaxes DNA, and this mechanism also accounts for the ability of gyrase to catenate and uncatenate DNA rings. Each round of supercoiling is driven by a conformational change induced by adenosine triphosphate (ATP) binding: ATP hydrolysis permits fresh cycles. The inhibition of gyrase by two classes of antimicrobials reflects its composition from two reversibly associated subunits. The A subunit is particularly associated with the concerted breakage-and-rejoining of DNA and the B subunit mediates energy transduction. Gyrase is a prototype for a growing class of prokaryotic and eukaryotic topoisomerases that interconvert complex forms by way of transient double-strand breaks.     

拟南芥减数分裂重组发生的遗传学研究

减数分裂是有性生殖物种世代交替的转折点,而减数分裂过程中发生的遗传重组则是遗传变异的源泉,并为有性生物的进化提供了推动力。现已发现许多基因在重组过程中起重要作用。由于重组蛋白的高度保守性,反向遗传学为研究植物重组蛋白的性质及作用提供了充分的证据。本文就近年来对模式植物拟南芥减数分裂中的DNA双链断裂形成与修复以及同源染色体重组交换等重要事件及其相关基因的功能进行了概述,尤其对ZMM家族蛋白在遗传重组中的作用进行了重点介绍。     

Distinct properties of the XY pseudoautosomal region crucial for male meiosis

Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11β isoform-specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.     

Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis

Meiotic recombination in budding yeast requires two RecA-related proteins, Rad51 and Dmc1, both of which form filaments on DNA capable of directing homology search and catalyzing formation of homologous joint molecules (JMs) and strand exchange. With use of a separation-of-function mutant form of Rad51 that retains filament-forming but not JM-forming activity, we show that the JM activity of Rad51 is fully dispensable for meiotic recombination. The corresponding mutation in Dmc1 causes a profound recombination defect, demonstrating Dmc1's JM activity alone is responsible for meiotic recombination. We further provide biochemical evidence that Rad51 acts with Mei5-Sae3 as a Dmc1 accessory factor. Thus, Rad51 is a multifunctional protein that catalyzes recombination directly in mitosis and indirectly, via Dmc1, during meiosis.     

植物减数分裂重组的影响因素·机制及在育种中的应用

总结了影响减数分裂重组频率的诸多因素,探讨了提高减数分裂重组频率对植物远缘杂交中不良基因与优异基因的不利连锁打破的可能,并就当前的问题和今后的发展前景进行了讨论。     

辐射作用下含修复DNA主链断裂随机动力学理论的研究

在辐射诱发DNA单链与双链断裂问题的研究中,迄今为止的主要理论——靶学说与击中理论,存在缺少修复机制等缺点,而Chadwick的理论仍属半经验性的.本文在Hug与Kellerer基本思想的基础上,发展了一个辐射诱发含修复DNA单、双链断裂的随机动力学理论,建立了随机动力学微分方程组数学模型.     

Involvement of mammalian Mus81 in genome integrity and tumor suppression

Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81-/- mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to gamma-irradiation. Remarkably, both homozygous Mus81-/- and heterozygous Mus81+/- mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.     

ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex

The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage.     

Structure of a NHEJ polymerase-mediated DNA synaptic complex

Nonhomologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway required to maintain genome stability. Many prokaryotes possess a minimalist NHEJ apparatus required to repair DSBs during stationary phase, composed of two conserved core proteins, Ku and ligase D (LigD). The crystal structure of Mycobacterium tuberculosis polymerase domain of LigD mediating the synapsis of two noncomplementary DNA ends revealed a variety of interactions, including microhomology base pairing, mismatched and flipped-out bases, and 3' termini forming hairpin-like ends. Biochemical and biophysical studies confirmed that polymerase-induced end synapsis also occurs in solution. We propose that this DNA synaptic structure reflects an intermediate bridging stage of the NHEJ process, before end processing and ligation, with both the polymerase and the DNA sequence playing pivotal roles in determining the sequential order of synapsis and remodeling before end joining.     

Pif1p helicase, a catalytic inhibitor of telomerase in yeast

Mutations in the yeast Saccharomyces cerevisiae PIF1 gene, which encodes a 5'-to-3' DNA helicase, cause telomere lengthening and a large increase in the formation rate of new telomeres. Here, we show that Pif1p acts by inhibiting telomerase rather than telomere-telomere recombination, and this inhibition requires the helicase activity of Pif1p. Overexpression of enzymatically active Pif1p causes telomere shortening. Thus, Pif1p is a catalytic inhibitor of telomerase-mediated telomere lengthening. Because Pif1p is associated with telomeric DNA in vivo, its effects on telomeres are likely direct. Pif1p-like helicases are found in diverse organisms, including humans. We propose that Pif1p-mediated inhibition of telomerase promotes genetic stability by suppressing telomerase-mediated healing of double-strand breaks.     

植物DNA双链断裂损伤修复机制研究进展

DNA双链断裂(DSBs)是细胞最严重的损伤形式之一。高等动植物中主要通过非同源末端连接（NHEJ）途径进行DNA双链断裂修复。该途径不依赖DNA同源性,由一些修复因子如：Ku蛋白异二聚体、DNA-PKcs 、XRCC4、ligaseⅣ等,将断裂末端直接连接进行修复。综述了植物DNA双链断裂损伤修复的主要途径及其相关基因研究的进展,探讨了植物DNA损伤修复研究中存在的问题与发展方向。     

Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations

Various types of chromosomal aberrations, including numerical (aneuploidy) and structural (e.g., translocations, deletions), are commonly found in human tumors and are linked to tumorigenesis. Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, whereas structural aberrations are caused by improperly repaired DNA breaks. Here, we demonstrate that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM, Chk2, and p53. We show that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Our data show that segregation errors can cause translocations and provide insights into the role of whole-chromosome instability in tumorigenesis.     

An oncogene-induced DNA damage model for cancer development

Of all types of DNA damage, DNA double-strand breaks (DSBs) pose the greatest challenge to cells. One might have, therefore, anticipated that a sizable number of DNA DSBs would be incompatible with cell proliferation. Yet recent experimental findings suggest that, in both precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn leads to formation of DNA DSBs. This continuous formation of DNA DSBs may contribute to the genomic instability that characterizes the vast majority of human cancers. In addition, in precancerous lesions, these DNA DSBs activate p53, which, by inducing apoptosis or senescence, raises a barrier to tumor progression. Breach of this barrier by various mechanisms, most notably by p53 mutations, that impair the DNA damage response pathway allows cancers to develop. Thus, oncogene-induced DNA damage may explain two key features of cancer: genomic instability and the high frequency of p53 mutations.     

DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7)

Faithful chromosome segregation and repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister-chromatid cohesion. Cohesion between sister chromatids is thought to be generated only during ongoing DNA replication by an obligate coupling between cohesion establishment factors such as Eco1 (Ctf7) and the replisome. Using budding yeast, we challenge this model by showing that cohesion is generated by an Eco1-dependent but replication-independent mechanism in response to DSBs in G(2)/M. Furthermore, our studies reveal that Eco1 has two functions: a cohesive activity and a conserved acetyltransferase activity, which triggers the generation of cohesion in response to the DSB and the DNA damage checkpoint. Finally, the DSB-induced cohesion is not limited to broken chromosomes but occurs also on unbroken chromosomes, suggesting that the DNA damage checkpoint through Eco1 provides genome-wide protection of chromosome integrity.