Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells

Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causes a wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects of DNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks. Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive to Cr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strand breaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important in repair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitive to Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage.     

DNA homologous recombination repair in mammalian cells

DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.     

Homologous recombination in a mammalian plasmid

Summary Bovine papillomavirus (BPV) shuttle vectors replicate as a circular plasmid in mouse cell nuclei without impairing host cell viability. We used these vectors to analyze homologous recombination in mammalian cells. When several BPV-based plasmids carrying direct repeats were introduced into C127 cells, we detected many recombinant plasmid molecules that have lost the sequence between the repeats. Many recombinant type molecules as well as parental type molecules were detected in all the cell clones isolated for analysis. Sequencing after rescue of the plasmid inEscherichia coli showed that most of the recombinants were from accurate homologous recombination. When the repeats on the plasmid were in inverted orientation, no crossing-over type products were detected. We discuss possible mechanisms that explain these features.     

Homologous recombination of adenovirus DNA in mammalian cells: enhanced recombination following UV-irradiation of the virus.

We have used adenovirus as a molecular probe to examine the recombination of viral DNA following infection of mammalian cells. The technique gives a quantitative measure of homologous recombination between adenovirus type 2 (Ad2) and Ad5PyMTR3. Ad5PyMTR3 is an insertion mutant of Ad5 containing polyoma virus (Py) DNA inserted into a deleted E1 region of the Ad5 genome. Cells were coinfected with Ad2 and Ad5PyMTR3 and at an appropriate time after infection, viral DNA was extracted from the infected cells, digested with restriction endonuclease and electrophoresed through an agarose gel. Although Ad2 and Ad5 have more than 99% DNA homology, they differ sufficiently in their restriction endonuclease patterns, such that recombinant viral DNA molecules containing the Py insert could be detected and quantified by Southern blotting and hybridization to a radioactive Py DNA probe. Using this method we are able to detect and quantitate recombinant viral DNA molecules containing the Py insert which are present at frequencies down to at least 1 in 100. Recombination was detected in Chinese hamster ovary cells, monkey kidney cells, human HeLa cells, normal human fibroblasts and SV40 transformed human fibroblasts. In experiments using HeLa cells, the frequency of recombination between the Py insert on Ad5PyMTR3 and a number of unique restriction enzyme sites on Ad2 increased with the distance from the Py insert to the restriction site. Also in HeLa cells, recombination increased with increasing amounts of viral DNA synthesis and with increasing UV dose to the virus. UV-irradiation of both coinfecting viruses with 1500 J/m2 resulted in a more than 100-fold reduction in the amount of viral DNA synthesized and about a 3-fold increase in the frequency of recombination.     

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.     

Phosphatidylinositol 5-phosphate biosynthesis is linked to PIKfyve and is involved in osmotic response pathway in mammalian cells

The cellular functions, regulation and enzymology of phosphatidylinositol (PtdIns) 5-P, the newest addition to the family of phosphoinositides (PI), are still elusive. Whereas a kinase that uses PtdIns-5-P as an intracellular substrate has been assigned, a kinase that produces it remained to be identified. Here we report that PIKfyve, the enzyme found to synthesize PtdIns-5-P in vitro and PtdIns-3,5-P(2) in vitro and in vivo, is responsible for PtdIns-5-P production in a cellular context. Evidence is based on examination of two groups of cell types by two independent approaches. First, [(32)P]orthophosphate-labeled cells (Sf9, 3T3-L1 fibroblasts, and 3T3-L1 adipocytes) that show a high pressure liquid chromatography (HPLC)-detectable peak of the PtdIns-5-P head group at basal conditions demonstrated a 20-50% increase in radioactive PtdIns-5-P amounts upon expression of PIKfyve(WT). Second, cell types (HEK293), in which the basal levels of radioactive PtdIns-5-P were undetectable by HPLC head group analysis, demonstrated higher in vitro type II PIP kinase-directed conversion of the endogenous PtdIns-5-P pool into PtdIns-4,5-P(2), when induced to express PIKfyve(WT). Conversely, a decrease by 60% in the conversion of PtdIns-5-P to PtdIns-4,5-P(2) was associated with induced expression of the dominant-negative kinase-deficient PIKfyve(K1831E) mutant in HEK293 cells. When 3T3-L1 fibroblasts and 3T3-L1 adipocytes were subjected to osmotic shock, levels of PtdIns-5-P measured by both approaches were found to decrease profoundly upon a hypo-osmotic stimulus. Together, these results identify PIKfyve as an enzyme responsible for PtdIns-5-P biosynthesis and indicate a role for PtdIns-5-P in osmotic response pathways in 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.     

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.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Homologous recombination in plants is organ specific

In this paper we analysed the genome stability of various Arabidopsis thaliana plant organs using a transgenic recombination system. The system was based on two copies of non-functional GUS (lines #651 and #11) or LUC (line #15D8) reporter genes serving as a recombination substrate. Both reporter assays showed that recombination in flowers or stems were rare events. Most of the recombination sectors were found in leaves and roots, with leaves having over 2-fold greater number of the recombination events per single cell genome as compared to roots. The recombination events per single genome were 9.7-fold more frequent on the lateral half of the leaves than on the medial halves. This correlated with a 2.5-fold higher metabolic activity in the energy source (lateral) versus energy sink (medial) of leaves. Higher metabolic activity was paralleled by a higher anthocyanin production in lateral halves. The level of double strand break (DSB) occurrence was also different among plant organs; the highest level was observed in roots and the lowest in leaves. High level of DSBs strongly positively correlated with the activity of the key repair enzymes, AtKU70 and AtRAD51. The ratio of AtRAD51 to AtKU70 expression was the highest in leaves, supporting the more active involvement of homologous recombination pathway in the repair of DSBs in this organ. Western blot analysis confirmed the real time PCR expression data for AtKU70 gene.     

Interchromosomal recombination is suppressed in mammalian somatic cells.

Homologous recombination occurs intrachromosomally as well as interchromosomally, both in mitotic (somatic) cells as well as meiotically in the germline. These different processes can serve very different purposes in maintaining the integrity of the organism and in enhancing diversity in the species. As shown here, comparison of the frequencies of intra- and interchromosomal recombination in meiotic and mitotic cells of both mouse and yeast argues that interchromosomal recombination is particularly low in mitotic cells of metazoan organisms. This result in turn suggests that the recombination machinery of metazoa might be organized to avoid the deleterious effects of homozygotization in somatic cells while still deriving the benefits of species diversification and of DNA repair.     

Homologous recombination is elevated in some Werner-like syndromes but not during normal in vitro or in vivo senescence of mammalian cells

Werner syndrome (WS) is a recessive genetic condition associated with markedly reduced replicative lifespans of cells in culture, high chromosomal instability in vivo and in vitro, and premature appearance of many characteristics of normal aging, including an increased incidence of cancer. We have monitored plasmid homologous recombination frequencies in diploid fibroblasts from 6 Werner or Werner-like syndrome patients, following transfection with a plasmid substrate containing 2 overlapping fragments of the TN5 Neor gene. Plasmid DNA recovered from these cells was then assayed for homologous recombination by (a) transformation of recA- bacteria to Ampr (indicating total viable plasmid) or Neor (indicating viable recombinant plasmid), and (b) by limited-cycle polymerase chain reaction (PCR) to co-amplify a recombinant fragment containing the overlap region, and a control region of the same plasmid, without bacterial transformation. Bacterial assay data indicated that recombination rates in 3 of the 6 WS strains were significantly elevated above normal controls; 4 of 6 appeared elevated by PCR assay. The highest-recombination WS strain showed evidence of reduced degradation of transfected plasmid DNA. For this small sample of WS strains, clinical severity of WS was not well correlated with recombination rate as determined by either assay (Pearson r = 0.78, not significant, for PCR assay); elevated recombination may, however, define a subset of WS at greatest risk for cancer and/or atherosclerosis. PCR assay of a hyperoxia-resistant HeLa cell line, displaying substantially increased chromosome breakage, indicated increased recombination between direct-repeat fragments. Nevertheless, elevated recombination in WS strains is unlikely to be secondary to impaired replicative capacity characteristic of WS cells, or to defective repair of chromosome damage which is increased in WS, since recombination in non-WS strains was unaffected by passage level or repeated UV irradiation.     

Homologous recombination in rat germline stem cells

Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.     

Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae

Genetic transformation of the Mucorales fungi has been problematic, since DNA transformed into the host rarely integrates and usually is mitotically unstable in the absence of selective pressure. In this study, transformation of Rhizopus oryzae was investigated to determine if the fate of introduced DNA could be predicted based on double-strand break repair and recombination mechanisms found in other fungi. A transformation system was developed with uracil auxotrophs of Rhizopus oryzae that could be complemented with the pyrG gene isolated in this work. DNA transformed as circular plasmids was maintained extrachromosomally in high-molecular-weight (>23 kb) concatenated arrangement. Type-I crossover integration into the pyrG locus and type-III pyrG gene replacement events occurred in approximately 1-5% of transformants. Linearization of the plasmid pPyr225 with a single restriction enzyme that cleaves within the vector sequence almost always resulted in isolates with replicating concatenated plasmids that had been repaired by end-joining recombination that restored the restriction site. The addition of a 40-bp direct repeat on either side of this cleavage site led to repair by homologous recombination between the repeated sequences on the plasmid, resulting in loss of the restriction site. When plasmid pPyr225 was digested with two different enzymes that cleave within the vector sequence to release the pyrG containing fragment, only pyrG gene replacement recombination occurred in transformants. Linearization of plasmid pPyr225 within the pyrG gene itself gave the highest percentage (20%) of type-I integration at the pyrG locus. However, end-joining repair and gene replacement events were still the predominant types of recombination found in transformations with this plasmid topology.     

Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair

Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.     

Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.