Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex

The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM (ataxia telangiectasia mutated). We demonstrate that MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. MRN makes multiple contacts with ATM and appears to stimulate ATM activity by facilitating the stable binding of substrates. Phosphorylation of Nbs1 is critical for MRN stimulation of ATM activity toward Chk2, but not p53. Kinase-deficient ATM inhibits wild-type ATM phosphorylation of Chk2, consistent with the dominant-negative effect of kinase-deficient ATM in vivo.     

ATM activation by oxidative stress

The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide-cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells.     

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.     

Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase

Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.     

Doubls-helical polynucleotides: immunochemical recognition of differing conformations

Rabbit antibodies to double-helical RNA react by complement fixation with synthetic or natural double-strand RNA but not with native DNA. In turn, human (from systemic lupus erythematosus patients) antibodies to native DNA do not react with double-strand RNA. Both types of antibodies show cross-reactions (from 1 percent to 50 percent) with RNA-DNA hybrids, but antibodies to the hybrids do not react at all with double-strand RNA or with native DNA. Antibodies to polydeoxyguanylate.polydeoxycytidylate also failed to react with native DNA.     

ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.     

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。     

ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage

The ataxia telangiectasia mutated (ATM) protein kinase is a critical component of a DNA-damage response network configured to maintain genomic integrity. The abundance of an essential downstream effecter of this pathway, the tumor suppressor protein p53, is tightly regulated by controlled degradation through COP1 and other E3 ubiquitin ligases, such as MDM2 and Pirh2; however, the signal transduction pathway that regulates the COP1-p53 axis following DNA damage remains enigmatic. We observed that in response to DNA damage, ATM phosphorylated COP1 on Ser(387) and stimulated a rapid autodegradation mechanism. Ionizing radiation triggered an ATM-dependent movement of COP1 from the nucleus to the cytoplasm, and ATM-dependent phosphorylation of COP1 on Ser(387) was both necessary and sufficient to disrupt the COP1-p53 complex and subsequently to abrogate the ubiquitination and degradation of p53. Furthermore, phosphorylation of COP1 on Ser(387) was required to permit p53 to become stabilized and to exert its tumor suppressor properties in response to DNA damage.     

Direct coupling between meiotic DNA replication and recombination initiation

During meiosis in Saccharomyces cerevisiae, DNA replication occurs 1. 5 to 2 hours before recombination initiates by DNA double-strand break formation. We show that replication and recombination initiation are directly linked. Blocking meiotic replication prevented double-strand break formation in a replication-checkpoint-independent manner, and delaying replication of a chromosome segment specifically delayed break formation in that segment. Consequently, the time between replication and break formation was held constant in all regions. We suggest that double-strand break formation occurs as part of a process initiated by DNA replication, which thus determines when meiotic recombination initiates on a regional rather than a cell-wide basis.     

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.     

Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage-induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.     

变铅青链霉菌DNA的研究——Ⅱ.位点特异性降解与DNA修饰的关系

变铅青链霉菌的DNA上存在着一种异常的修饰,使其在含有微量Fe~(++)的缓冲液中电泳时,双链DNA遭到降解。DNA的切割是位点特异性的。与已知修饰特征的DNA进行同步试验发现,变铅青链霉菌的这种特异性修饰与目前所发现的修饰系统(如DNA甲基化)均不相同,很可能是一种新的修饰系统。     

p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2

Although broken chromosomes can induce apoptosis, natural chromosome ends (telomeres) do not trigger this response. It is shown that this suppression of apoptosis involves the telomeric-repeat binding factor 2 (TRF2). Inhibition of TRF2 resulted in apoptosis in a subset of mammalian cell types. The response was mediated by p53 and the ATM (ataxia telangiectasia mutated) kinase, consistent with activation of a DNA damage checkpoint. Apoptosis was not due to rupture of dicentric chromosomes formed by end-to-end fusion, indicating that telomeres lacking TRF2 directly signal apoptosis, possibly because they resemble damaged DNA. Thus, in some cells, telomere shortening may signal cell death rather than senescence.     

多穗轴节类小麦性状关系探讨

研究多穗轴节类小麦资源的穗轴节数与小穗数间及它们与其它穗部性状和抽穗间的关系是评价多穗轴节类利用价值,确定利用方式和策略的重要基础。本文通过对部分多穗轴节类和普通穗类基因型及多穗轴节×普通穗类杂种Ｆ１性状间简单相关和多元相关分析发现：１．多穗轴节类所具有的不利性状是可以克服的,只是不同性状改良难易程度不同。因而在多穗轴节小麦育种中应进一步创造、引入变异类型,丰富遗传资源,使之更接近早熟高产多穗轴节育种目标。２．各穗部性状在（ＭＲＮ×ＣＳ）Ｆ１中有利和不利关系共同存在。以现有多穗轴节类为亲本利用杂种优势,需要综合考虑性状间的关系和特定的生态环境要求。３．本试验采用的多穗轴节类基因型的多穗轴节性状和小穗数性状可能存在不同的遗传基础,两者与其它穗部性状和生育期间的关系也有所不同,推测其差异可能是由于穗轴节和结实小穗的发育差异造成的。在这些多穗轴节类基因型的基础上进行多穗轴节小麦育种应注意区别穗轴节数和结实小穗数,在穗轴节数多的基础上进一步对结实小穗数进行选择。     

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.     

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.     

Activation of the cellular DNA damage response in the absence of DNA lesions

The cellular DNA damage response (DDR) is initiated by the rapid recruitment of repair factors to the site of DNA damage to form a multiprotein repair complex. How the repair complex senses damaged DNA and then activates the DDR is not well understood. We show that prolonged binding of DNA repair factors to chromatin can elicit the DDR in an ATM (ataxia telangiectasia mutated)- and DNAPK (DNA-dependent protein kinase)-dependent manner in the absence of DNA damage. Targeting of single repair factors to chromatin revealed a hierarchy of protein interactions within the repair complex and suggests amplification of the damage signal. We conclude that activation of the DDR does not require DNA damage and stable association of repair factors with chromatin is likely a critical step in triggering, amplifying, and maintaining the DDR signal.     

Identification of a DNA nonhomologous end-joining complex in bacteria

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.     

Transforming Activity in Both Complementary Strands of Bacillus subtilis DNA

Under conditions favoring single-strand transformation, the two complementary strands of Bacillus subtilis DNA, separated by differential complexing with polyriboguanylic acid, have identical transforming activity. Moreover separated single strands, upon renaturation with unmarked (recipient) DNA, form heteroduplex molecules with similar double-strand transformin activity. These findings bear upon the mechanism of DNA integration.