BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.     

Differential roles of XRCC2 in homologous recombinational repair of stalled replication forks

Homologous recombination is an important mechanism in DNA replication to ensure faithful DNA synthesis and genomic stability. In this study, we investigated the role of XRCC2, a member of the RAD51 paralog family, in cellular recovery from replication arrest via homologous recombination. The protein expression of XRCC2, as well as its binding partner RAD51D, is dramatically increased in S- and G2-phases, suggesting that these proteins function during and after DNA synthesis. XRCC2 mutant irs1 cells exhibit hypersensitivity to hydroxyurea (HU) and are defective in the induction of RAD51 foci after HU treatment. In addition, the HU-induced chromatin association of RAD51 is deficient in irs1 mutant. Interestingly, irs1 cells are only slightly sensitive to thymidine and able to form intact RAD51 foci in S-phase cells arrested with thymidine. Irs1 cells showed increased level of chromatin-bound RAD51 as well as the wild type cells after thymidine treatment. Both HU and thymidine induce gamma-H2AX foci in arrested S-phase nuclei. These results suggest that XRCC2 is involved in repair of HU-induced damage, but not thymidine-induced damage, at the stalled replication forks. Our data suggest that there are at least two sub-pathways in homologous recombination, XRCC2-dependent and -independent, for repair of stalled replication forks and assembly of RAD51 foci following replication arrest in S-phase.     

DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N²-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N²-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.     

DNA damage discrimination at stalled replication forks by the Rad5 homologs HLTF and SHPRH

In this issue of Molecular Cell, Lin et al. (2011) describe how HLTF and SHPRH, the human homologs of yeast Rad5, can discriminate between MMS-induced versus UV-induced DNA damage. The results have important implications for the suppression of damage-specific mutagenesis and for the maintenance of genomic stability.     

The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair

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An SMC-like protein is required for efficient homologous recombination in Arabidopsis.

In plants, the observed low frequency of gene targeting and intrachromosomal recombination contrasts markedly with the efficient extrachromosomal recombination of DNA. Thus, chromatin accessibility can have a major influence on the recombination frequency of chromosomal DNA in vivo. An Arabidopsis mutant hypersensitive to a range of DNA-damaging treatments (UV-C, X-rays, methyl methanesulfonate and mitomycin C) is also defective in somatic intrachromosomal homologous recombination. The wild-type gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family involved in structural changes in chromosomes. Although loss of SMC function is lethal in other eukaryotes, growth of the Arabidopsis mutant is normal in the absence of genotoxic treatments. This suggests a surprisingly specialized function for this protein in plants, and provides the first in vivo evidence for the involvement of an SMC protein in recombinational DNA repair. It is possible that SMC-like proteins in plants alleviate suppressive chromatin structure limiting homologous recombination in somatic cells.     

BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells

Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 µM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers.     

PARP is activated at stalled forks to mediate Mre11‐dependent replication restart and recombination

If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP‐ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea‐induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.     

XRCC3 is required for efficient repair of chromosome breaks by homologous recombination

XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.     

Rad52 sumoylation and its involvement in the efficient induction of homologous recombination

The protein Rad52 is a key player in various types of homologous recombination and is essential to maintenance of genomic integrity. Although evidence indicates that Rad52 is modified by SUMO, the physiological relevance of this sumoylation remains unclear. Here, we identify the conditions under which Rad52 sumoylation is induced, and clarify the role of this modification in homologous recombination. Oligomerization of Rad52 was a prerequisite for sumoylation, and the modification occurred in the cell proceeding S phase being exposed to the DNA-damaging agent methyl methanesulfonate (MMS). Following exposure to MMS, sumoylated Rad52 accumulated in rad51 cells, but not in the recombination-related gene mutants, rad54, rad55, rad59, sgs1, or srs2. The accumulation of sumoylated Rad52 was suppressed in rad51 cells expressing Rad51-K191R, an ATPase-defective protein presumed to be recruited to ssDNA. Although the sumoylation defective mutant rad52-3KR (K10R/K11R/K220R) showed no defect in mating-type switching, which did not lead to Rad52 sumoylation in wild-type cells, the mutant did demonstrate a partial defect in MMS-induced interchromosomal homologous recombination.     

Replisome assembly and the direct restart of stalled replication forks

Failure to reactivate either stalled or collapsed replication forks is a source of genomic instability in both prokaryotes and eukaryotes. In prokaryotes, dedicated fork repair systems that involve both recombination and replication proteins have been identified genetically and characterized biochemically. Replication conflicts are solved through several pathways, some of which require recombination and some of which operate directly at the stalled fork. Some recent biochemical observations support models of direct fork repair in which the removal of the blocking template lesion is not always required for replication restart.     

Rad54, the motor of homologous recombination

Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions.     

The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.     

DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylation

Rad18 protein is required for mono-ubiquitination of PCNA and trans-lesion synthesis during DNA lesion bypass in eukaryotic cells but it remains unknown how it is activated after DNA damage. We expressed GFP-tagged human (h)Rad18 in Chinese hamster cells and found that it can be completely extracted from undamaged nuclei by Triton X-100 and methanol. However, several hours after treatment with methyl methanesulfonate (MMS) Triton-insoluble form of GFP-hRad18 accumulates in S-phase nuclei where it colocalizes with PCNA. This accumulation is suppressed by inhibitors of protein kinases staurosporine and wortmannin but is not effected by roscovitine. We also found that methyl methanesulfonate induces phosphorylation of Ser-317 in protein kinase Chk1 and Ser-139 in histone H2AX and stimulates formation of single-stranded DNA at replication foci. Together, our results suggest that MMS-induced accumulation of hRad18 protein at stalled forks involves protein phosphorylation which may be performed by S-phase checkpoint kinases.     

Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival

PriA, a 3′ → 5′ superfamily 2 DNA helicase, acts to remodel stalled replication forks and as a specificity factor for origin-independent assembly of a new replisome at the stalled fork. The ability of PriA to initiate replication at stalled forked structures ensures complete genome replication and helps to protect the cell from illegitimate recombination events. This review focuses on the activities of PriA and its role in replication fork assembly and maintaining genomic integrity.     

Phosphorylation of human Rad9 is required for genotoxin-activated checkpoint signaling

Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in genotoxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9-/- mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HU- and UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9-/- cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.     

Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing

Stable expression of Rad51 siRNA was used to generate mouse hybridoma cell lines in which endogenous Rad51 levels were depleted by as much as 60%. Stable Rad51 knockdowns feature reduced homologous recombination responses. The relative ease with which stable Rad51 knockdowns were recovered was surprising, given the embryonic lethality of Rad51 ablation. Interestingly, Rad51-depleted hybridoma cell lines are characterized by reduced levels of p53 protein. Completely unexpected, was the finding that Rad51-depleted hybridoma cell lines are also reduced for the breast cancer susceptibility 2 (Brca2) protein. Additionally, hybridoma cell lines that are siRNA depleted for mouse Brca2 show a corresponding reduction in Rad51 and p53 proteins. Furthermore, cellular levels of Rad51, Brca2 and p53 can be elevated in these cell lines by ectopic expression of wild-type human Rad51 and wild-type human BRCA2. In marked contrast, hybridoma cell lines that are siRNA depleted for mouse p53 feature relatively normal Rad51 and Brca2 levels. These results suggest that cellular levels of Brca2 and Rad51 are mutually dependent on each other, and that low levels of these proteins provide selective pressure for reduction of p53, which permits cell growth.     

Brca2, Rad51 and Mre11: performing balancing acts on replication forks

Homologous recombination (HR) is required for faithful repair of double strand breaks (DSBs) and is believed to be important for DNA replication under stressful conditions in unicellular organisms. However, its role during DNA replication in high eukaryotes has always been elusive. In particular, due to the essential nature of its main players it has been difficult to dissect the direct role of HR in DNA replication. Recent studies revealed that some key HR factors such as Rad51 and BRCA2 play unexpected functions during DNA replication by protecting nascent DNA from Mre11 mediated degradation, which takes place at stalled replication forks. These novel functions appear to be essential to ensure smooth progression of DNA replication and to promote maintenance of genome stability.