Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy

BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.     

Binding of double-strand breaks in DNA by human Rad52 protein

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.     

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

Transgeneration memory of stress in plants

Owing to their sessile nature, plants are constantly exposed to a multitude of environmental stresses to which they react with a battery of responses. The result is plant tolerance to conditions such as excessive or inadequate light, water, salt and temperature, and resistance to pathogens. Not only is plant physiology known to change under abiotic or biotic stress, but changes in the genome have also been identified. However, it was not determined whether plants from successive generations of the original, stressed plants inherited the capacity for genomic change. Here we show that in Arabidopsis thaliana plants treated with short-wavelength radiation (ultraviolet-C) or flagellin (an elicitor of plant defences), somatic homologous recombination of a transgenic reporter is increased in the treated population and these increased levels of homologous recombination persist in the subsequent, untreated generations. The epigenetic trait of enhanced homologous recombination could be transmitted through both the maternal and the paternal crossing partner, and proved to be dominant. The increase of the hyper-recombination state in generations subsequent to the treated generation was independent of the presence of the transgenic allele (the recombination substrate under consideration) in the treated plant. We conclude that environmental factors lead to increased genomic flexibility even in successive, untreated generations, and may increase the potential for adaptation.     

Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination.

The repair of DNA double-strand breaks is essential for cells to maintain their genomic integrity. Two major mechanisms are responsible for repairing these breaks in mammalian cells, non-homologous end-joining (NHEJ) and homologous recombination (HR): the importance of the former in mammalian cells is well established, whereas the role of the latter is just emerging. Homologous recombination is presumably promoted by an evolutionarily conserved group of genes termed the Rad52 epistasis group. An essential component of the HR pathway is the strand-exchange protein, known as RecA in bacteria or Rad51 in yeast. Several mammalian genes have been implicated in repair by homologous recombination on the basis of their sequence homology to yeast Rad51: one of these is human XRCC2. Here we show that XRCC2 is essential for the efficient repair of DNA double-strand breaks by homologous recombination between sister chromatids. We find that hamster cells deficient in XRCC2 show more than a 100-fold decrease in HR induced by double-strand breaks compared with the parental cell line. This defect is corrected to almost wild-type levels by transient transfection with a plasmid expressing XRCC2. The repair defect in XRCC2 mutant cells appears to be restricted to recombinational repair because NHEJ is normal. We conclude that XRCC2 is involved in the repair of DNA double-strand breaks by homologous recombination.     

Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads approximately 40 kb in either direction from the DSB. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX is required for histone loss, which also depends on INO80, a nucleosome remodelling complex. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage.     

Arctic microorganisms respond more to elevated UV-B radiation than CO2

Surface ultraviolet-B radiation and atmospheric CO2 concentrations have increased as a result of ozone depletion and burning of fossil fuels. The effects are likely to be most apparent in polar regions where ozone holes have developed and ecosystems are particularly sensitive to disturbance. Polar plant communities are dependent on nutrient cycling by soil microorganisms, which represent a significant and highly labile portion of soil carbon (C) and nitrogen (N). It was thought that the soil microbial biomass was unlikely to be affected by exposure of their associated plant communities to increased UV-B. In contrast, increasing atmospheric CO2 concentrations were thought to have a strong effect as a result of greater below-ground C allocation. In addition, there is a growing belief that ozone depletion is of only minor environmental concern because the impacts of UV-B radiation on plant communities are often very subtle. Here we show that 5 years of exposure of a subarctic heath to enhanced UV-B radiation both alone and in combination with elevated CO2 resulted in significant changes in the C:N ratio and in the bacterial community structure of the soil microbial biomass.     

UV-B和NO对胞壁蛋白的影响

通过对玉米幼苗叶片细胞壁中蛋白组分、质量分数和过氧化物酶活性的分析检测,证明UV-B光胁迫下,内、外源NO作为UV-B辐射光氧化胁迫的信使负责胁迫信号的转导,激活细胞壁过氧化物酶的活性,致使细胞壁共价键结合蛋白,积累,从而钝化许多功能蛋白质的活力.而没有UV-B光胁迫时,适量的内、外源NO是抑制细胞壁过氧化物酶活性的有效抗胁迫分子,保证细胞壁中足量离子结合和游离蛋白质量分数,维持细胞壁中活跃的代谢功能.因此,在细胞壁中,一氧化氮能够介导UV-B辐射对细胞壁中结构和功能蛋白质的影响,为转导增强UV-B光胁迫的信号分子.     

Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.     

增强UV-B辐射对高等植物的作用以及植物的适应

综述了增强的UV-B辐射对植物的影响,植物的适应机制,以及损伤、适应和修复三者之间 的平衡关系.     

Template switching during break-induced replication

DNA double-strand breaks (DSBs) are potentially lethal lesions that arise spontaneously during normal cellular metabolism, as a consequence of environmental genotoxins or radiation, or during programmed recombination processes. Repair of DSBs by homologous recombination generally occurs by gene conversion resulting from transfer of information from an intact donor duplex to both ends of the break site of the broken chromosome. In mitotic cells, gene conversion is rarely associated with reciprocal exchange and thus limits loss of heterozygosity for markers downstream of the site of repair and restricts potentially deleterious chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). BIR from one of the two ends of a DSB would result in loss of heterozygosity, suggesting that BIR is suppressed when DSBs have two ends so that repair occurs by the more conservative gene conversion mechanism. Here we show that BIR can occur by several rounds of strand invasion, DNA synthesis and dissociation. We further show that chromosome rearrangements can occur during BIR if dissociation and reinvasion occur within dispersed repeated sequences. This dynamic process could function to promote gene conversion by capture of the displaced invading strand at two-ended DSBs to prevent BIR.     

CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair

Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumorigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination. Here we show that the carboxy-terminal region of BRCA2, which interacts directly with the essential recombination protein RAD51, contains a site (serine 3291; S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 is low in S phase when recombination is active, but increases as cells progress towards mitosis. This modification blocks C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by decreasing S3291 phosphorylation and stimulating interactions with RAD51. These results indicate that S3291 phosphorylation might provide a molecular switch to regulate RAD51 recombination activity, providing new insight into why BRCA2 C-terminal deletions lead to radiation sensitivity and cancer predisposition.     

He-Ne激光和增强UV-B辐射对小麦幼苗氮代谢的影响

为研究He-Ne激光对增强UV-B辐射后损伤的修复作用,采用5 mW.mm-2He-Ne激光辐照、10.08 kJ.m2.d1的UV-B辐射及二者组合对舜麦1718号小麦幼苗进行处理,测定各处理组小麦幼苗硝酸还原酶活性、一氧化氮合成酶含量和膜透性的变化.分析结果显示:He-Ne激光辐照可使UV-B辐射后小麦幼苗硝酸还原酶活力升高1.2倍,显著改善了增强UV-B辐射后硝酸还原酶活性降低的程度;而一氧化氮合成酶(NOS)含量B组和BL组分别高于CK组45.1%和17.8%,现促进作用.     

Human CtIP promotes DNA end resection

In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.     

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.     

Functional link of BRCA1 and ataxia telangiectasia gene product in DNA damage response

BRCA1 encodes a familial breast cancer suppressor that has a critical role in cellular responses to DNA damage. Mouse cells deficient for Brca1 show genetic instability, defective G2-M checkpoint control and reduced homologous recombination. BRCA1 also directly interacts with proteins of the DNA repair machinery and regulates expression of both the p21 and GADD45 genes. However, it remains unclear how DNA damage signals are transmitted to modulate the repair function of BRCA1. Here we show that the BRCA1-associated protein CtIP becomes hyperphosphorylated and dissociated from BRCA1 upon ionizing radiation. This phosphorylation event requires the protein kinase (ATM) that is mutated in the disease ataxia telangiectasia. ATM phosphorylates CtIP at serine residues 664 and 745, and mutation of these sites to alanine abrogates the dissociation of BRCA1 from CtIP, resulting in persistent repression of BRCA1-dependent induction of GADD45 upon ionizing radiation. We conclude that ATM, by phosphorylating CtIP upon ionizing radiation, may modulate BRCA1-mediated regulation of the DNA damage-response GADD45 gene, thus providing a potential link between ATM deficiency and breast cancer.     

Rad54 protein promotes branch migration of Holliday junctions

Homologous recombination has a crucial function in the repair of DNA double-strand breaks and in faithful chromosome segregation. The mechanism of homologous recombination involves the search for homology and invasion of the ends of a broken DNA molecule into homologous duplex DNA to form a cross-stranded structure, a Holliday junction (HJ). A HJ is able to undergo branch migration along DNA, generating increasing or decreasing lengths of heteroduplex. In both prokaryotes and eukaryotes, the physical evidence for HJs, the key intermediate in homologous recombination, was provided by electron microscopy. In bacteria there are specialized enzymes that promote branch migration of HJs. However, in eukaryotes the identity of homologous recombination branch-migration protein(s) has remained elusive. Here we show that Rad54, a Swi2/Snf2 protein, binds HJ-like structures with high specificity and promotes their bidirectional branch migration in an ATPase-dependent manner. The activity seemed to be conserved in human and yeast Rad54 orthologues. In vitro, Rad54 has been shown to stimulate DNA pairing of Rad51, a key homologous recombination protein. However, genetic data indicate that Rad54 protein might also act at later stages of homologous recombination, after Rad51 (ref. 13). Novel DNA branch-migration activity is fully consistent with this late homologous recombination function of Rad54 protein.     

Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55-Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55-Rad57 heterodimer associates with the Rad51-single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51-Rad55-Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.