RAD51调控REV1参与DNA双链断裂修复

REV1是跨损伤聚合酶Y家族的重要成员之一,它不仅作为支架蛋白介导Y家族聚合酶招募至损伤位点完成跨损伤DNA合成(translesion DNA synthesis, TLS),还可利用自身的dCMP转移酶活性在一些损伤位点对侧整合dCMP参与TLS。此外,REV1也被报导参与调控同源重组修复。为进一步探讨REV1互作蛋白RAD51和RAD51C在其参与的同源重组修复通路中的调控作用,本研究采用脉冲氮激光微辐射实验,发现RAD51可调控REV1到双链断裂位点的募集。同时,免疫荧光实验结果证明REV1也反过来影响RAD51应答CPT损伤。然而敲低RAD51C并不影响REV1到DNA双链断裂位点的招募。结果表明,REV1和RAD51在HR通路中存在彼此相互调控的关系。     

靶向Rad51与肿瘤治疗的研究进展

DNA损伤反应在维持细胞基因组稳定性和机体存活发挥重要作用。DNA双链断裂(Double strand breaks,DSBs)是DNA损伤最严重的形式。同源重组修复是体内参与DSBs损伤修复的重要机制之一,其中Rad51是体内参与同源重组性DNA修复的关键因子。Rad51在人类的多种肿瘤组织中高表达,如乳腺癌、非小细胞肺癌、前列腺癌等,与肿瘤的转移和恶化相关。如何有效下调肿瘤组织中的Rad51的水平,降低肿瘤细胞的DNA损伤修复能力,从而提高肿瘤治疗的疗效具有潜在的临床应用价值。本文对近年来的一个研究热点靶向Rad51在肿瘤治疗研究中的应用进行综述。     

DNA End Resection:Facts and Mechanisms

DNA double-strand breaks (DSBs), which arise following exposure to a number of endogenous and exogenous agents, can be repaired by either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways in eukaryotic cells. A vital step in HR repair is DNA end resection, which generates a long 30 single-stranded DNA (ssDNA) tail that can invade the homologous DNA strand. The generation of 30 ssDNA is not only essential for HR repair, but also promotes activation of the ataxia telangiectasia and Rad3-related protein (ATR). Multiple fac-tors, including the MRN/X complex, C-terminal-binding protein interacting protein (CtIP)/Sae2, exonuclease 1 (EXO1), Bloom syndrome protein (BLM)/Sgs1, DNA2 nuclease/helicase, and several chromatin remodelers, cooperate to complete the process of end resection. Here we review the basic machinery involved in DNA end resection in eukaryotic cells.     

A comparison of calcium phosphate coprecipitation and electroporation

Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.     

RAD51 is involved in repair of damage associated with DNA replication in mammalian cells

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.     

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.     

Genome instability and embryonic developmental defects in RMI1 deficient mice

RMI1 forms an evolutionarily conserved complex with BLM/TOP3α/RMI2 (BTR complex) to prevent and resolve aberrant recombination products, thereby promoting genome stability. Most of our knowledge about RMI1 function has been obtained from biochemical studies in vitro. In contrast, the role of RMI1 in vivo remains unclear. Previous attempts to generate an Rmi1 knockout mouse line resulted in pre-implantation embryonic lethality, precluding the use of mouse embryonic fibroblasts (MEFs) and embryonic morphology to assess the role of RMI1 in vivo. Here, we report the generation of an Rmi1 deficient mouse line (hy/hy) that develops until 9.5 days post coitum (dpc) with marked defects in development. MEFs derived from Rmi1^hy/hy are characterized by severely impaired cell proliferation, frequently having elevated DNA content, high numbers of micronuclei and an elevated percentage of partial condensed chromosomes. Our results demonstrate the importance of RMI1 in maintaining genome integrity and normal embryonic development.     

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.     

Targeting the homologous recombination pathway by small molecule modulators

During the last decade, the use of small molecule (MW <500 Da) compounds that modulate (inhibit or activate) important proteins of different biological pathways became widespread. Recently, the homologous recombination (HR) pathway emerged as a target for such modulators. Development of small molecule modulators pursues two distinct but not mutually exclusive purposes: to create a research tool to study the activities or functions of proteins of interest and to produce drugs targeting specific pathologies. Here, we review the progress of small molecule development in the area of HR.