Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.     

Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability

Our recent work has shown that activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. We have extended these observations by investigating two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, which are known to be regulated by mitogen stimulation. We now show that the phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation.     

同源重组修复与基因组不稳定性

同源重组修复途径极为精确,对于维持基因组的稳定性和完整性至关重要.因此,同源重组修复途径的功能障碍通常会导致严重的基因组不稳定,从而促进肿瘤的发生和演进;但这也可能破坏细胞固有的代谢过程,而成为治疗肿瘤耐药的关键靶点.本文将对同源重组修复的过程、类型及同源重组功能障碍与基因组不稳定性之间的关系加以综述.     

Polymorphisms in nonhomologous end-joining genes associated with breast cancer risk and chromosomal radiosensitivity

As enhanced chromosomal radiosensitivity (CRS) results from non- or misrepaired double strand breaks (DSBs) and is a hallmark for breast cancer and single nucleotide polymorphisms (SNPs) in DSB repair genes, such as non homologous end-joining (NHEJ) genes, could be involved in CRS and genetic predisposition to breast cancer. In this study, we investigated the association of five SNPs in three different NHEJ genes with breast cancer in a population-based case-control setting. The total patient population composed of a selected group of patients with a family history of the disease and an unselected group, consisting mainly of sporadic cases. SNP analysis showed that the c.2099-2408G>A SNP (XRCC5Ku80) [corrected] has a significant, positive odds ratio (OR) of 2.81 (95% confidence interval (CI): 1.30-6.05) for the heterozygous (He) and homozygous variant (HV) genotypes in the selected patient group. For the c.-1310 C>G SNP (XRCC6Ku70)[corrected] a significant OR of 1.85 (95%CI: 1.01-3.41) was found for the He genotype in the unselected patient group. On the contrary, the HV genotype of c.1781G>T (XRCC6Ku70) [corrected] displays a significant, negative OR of 0.43 (95%CI: 0.18-0.99) in the total patient population. The He+HV genotypes of the c.2099-2408G>A SNP (XRCC5Ku80) [corrected] also showed high and significant ORs in the group of "radiosensitive," familial breast cancer patients. In conclusion, our results provide preliminary evidence that the variant allele of c.-1310C>G (XRCC6Ku70) [corrected]and c.2099-2408G>A (XRCC5Ku80) [corrected] are risk alleles for breast cancer as well as CRS. The HV genotype of c.1781G>T (XRCC6Ku70) [corrected] on the contrary, seems to protect against breast cancer and ionizing radiation induced micronuclei.     

Homologous recombination and dynamics of rhizobial genomes

The nitrogen-fixing bacteria commonly known as rhizobia are attractive organisms due to their symbiotic association with legume plants. Their genomes contain a large number of redundant genetic elements. These reiterations might participate in homologous recombination events and lead to diverse genomic rearrangements. Here we analyze the role of homologous recombination in the dynamics of these bacterial genomes, as well as its possible biological consequences.     

Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells

The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation. This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining). We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining. In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site. In the other one, the DSB is made by the RAG1 and RAG2 proteins. After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion. We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency. V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use. Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination. These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways.     

Differential expression of DNA nonhomologous end-joining proteins Ku70 and Ku80 in melanoma progression.

Ku70 and Ku80 heterodimers function as regulatory subunits of the DNA-dependent protein kinase and play a very important role in the repairing of DNA double-strand breaks. Although Ku70 is proposed as a candidate for a tumor suppressor gene, not many data are available on Ku70 and Ku80 expression in human tumors. The main aim of this study was to investigate the expression of Ku70 and Ku80 in the ultraviolet-induced lesions-nevus cell nevi, lentigos maligna, and malignant melanomas. Nineteen nevus cell nevi, 23 lentigos maligna, 76 primary melanomas, and 31 melanoma metastases were stained immunohistochemically for the presence of Ku70 and Ku80 proteins. Ku70 and Ku80 expression was preserved in about 80% of nevi, 26% of lentigo maligna, 45% of primary melanomas, and 67% of melanoma metastases. Highly significant differences in Ku70 and Ku80 expression were found between nevi, lentigo maligna, and melanomas. In Cox regression, Ku70 and Ku80 were shown to be highly significant influences on patients' prognosis. Significant correlations between Ku70 and Ku80 expressions were found in nevi, lentigo maligna, and primary melanomas. These correlations were not more present in melanoma metastases. To summarize, earlier phases of melanoma progression seem to be connected with the loss of expression of Ku proteins. Metastatic spread is related to dysregulation of the Ku70 and Ku80 axis.     

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.     

Homologous recombination evidence in human and swine influenza A viruses

Dynamic gene mutation and the reassortment of genes have been considered as the key factors responsible for influenza A virus virulence and host tropism change. This study reports several significant evidence demonstrating that homologous recombination also takes place between influenza A viruses in human and swine lineages. Moreover, in a mosaic descended from swine H1N1 subtype and human H2N2, we found that its minor putative parent might be a derivative from the human cold-adapted vaccine lineage, which suggests that live vaccine is capable of playing a role in genetic change of influenza A virus via recombination with circulating viruses. These results would be important for knowing the molecular mechanism of mammal influenza A virus heredity and evolution.     

Effects of DNA topoisomerase inhibitors on nonhomologous and homologous recombination in mammalian cells

To study the involvement of DNA topoisomerases in recombination in mammalian cells, we used gene transfer assays to examine the effects of DNA topoisomerase inhibitors on nonhomologous (illegitimate) and homologous recombination. The assays were performed by transfecting adenine phosphoribosyltransferase-deficient (APRT⁻) CHO cells with plasmids carrying the wild-type or mutant aprt genes and by treating the cells with the inhibitors, followed by subsequent cultivation to select for APRT-positive (APRT⁺) colonies. Treatments with DNA topoisomerase II inhibitors such as VP-16, VM-26, ICRF-193 resulted in a 3- to 5-fold stimulation of integration of both closed-circular and linearized plasmids carrying the wild-type aprt gene into the recipient genome through nonhomologous recombination. The same treatments also increased 6- to 9-fold and 3-fold the number of APRT⁺ recombinant colonies that were generated by cotransfecting two closed-circular plasmids with nonoverlapping defective aprt genes and their linearized equivalents, respectively. However, this cotransfection assay involved intrinsically nonhomologous recombination processes; normalization of the frequencies by dividing them with those of the above nonhomologous recombination revealed 2-fold enhancement of homologous recombination events between the circular mutant genes but not between the linear ones. In contrast, DNA topoisomerase I inhibitor, camptothecin, showed no such effect on either recombination. From these results, we discuss the function of DNA topoisomerases on recombination in mammalian cells.     

Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid-chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.     

Common fragile site FRA11G and rare fragile site FRA11B at 11q23.3 encompass distinct genomic regions

Fragile sites are specific genomic loci that are particularly prone to chromosomal breakage. Based on their incidence in the human population, they are divided into rare fragile sites occurring in less than 5% of all individuals and common fragile sites being a constitutional feature of the genome of probably all individuals. In this study, cloning of unstable DNA sequences, which have been previously genetically tagged with a marker gene, was the basis for defining the genomic localization of the common fragile site FRA11G at 11q23.3. Mapping of the fragile site with six-color fluorescence in situ hybridization (FISH) resulted in the precise genomic localization of FRA11G to a 4.5 Mb region. The chromosomal subband 11q23.3 harbors both the common fragile site FRA11G and the rare fragile site FRA11B. Here, we show that FRA11G maps 0.8 Mb proximal to the genomic region previously defined to be affected by expression of FRA11B; thus, the common and the rare fragile sites at 11q23.3 encompass distinct genomic regions. The region of FRA11G is known to be involved in somatic and germline recurrent aberrations, and it is conceivable that genetic damage resulting from this fragile site might contribute to clinical phenotypes.     

Coincidence in fragile site expression with fluorodeoxyuridine and bromodeoxyuridine

Fragile site expression induced by 10 micrograms/ml or 20 micrograms/ml fluorodeoxyuridine (FudR) and 25 micrograms/ml or 50 micrograms/ml bromodeoxyuridine (BrdU) was studied in lymphocyte cultures of six healthy individuals. A significant decrease in mitotic indexes in respect to control cultures was observed with both FudR concentrations used. The cells showing chromosome aberrations and the total number of cytogenetic alterations were significantly increased both in FudR (p less than 0.001) and BrdU (25 micrograms/ml) (p less than 0.05) treated cultures with respect to the control culture. A site showing a gap or a break was defined as fragile if it appeared in 1% of the cells analyzed and in at least three of the six individuals studied with the same culture treatment. Using these criteria, fragile sites 4q31, 5q15, 6p22, 7p13, 7q32, 13q21, and 14q24 were induced in different proportions by both chemical agents. Although these drugs act via different mechanisms, they both substitute for thymidine in DNA. Our findings suggest that FudR is a more potent common fragile site inducer than BrdU.     

Frequency and stability of the fragile X premutation

Although considered the most common heritable cause of neurodevelopmentaldisability, precise prevalence figures for the FMR1 mutationin the general population are lacking. Since no fragile X premutationalleles have yet been observed to originate from FMR1 alleleswithin the normal size range, there is also little Informationavailable about the origin of the fragile X premutation andmechanisms leading to instability of the FMR1 trinucleotiderepeat region. In this study, 977 genetically unrelated individualsfrom families unselected for mental retardation or fragile Xwere analyzed with Southern blot analysis for the presence ofFMR1 mutations. A subgroup of subjects with evidence of a largeCGG repeat number, and any available relatives, were furtherstudied with PCR to investigate the stability of the trinucleotiderepeat segment of FMR1. One subject had a 75 repeat length whichwas unstable (increased In size) when passed to subsequent generations.This Includes one male descendent who had a premutatlon/fullmutation mosaic pattern. Two other alleles with     

Homologous recombination is not enhanced in UV-irradiated normal and repair-deficient human fibroblasts

Many mammalian cells exhibit damage-inducible phenomena that resemble the bacterial SOS functions. However, whereas RecA plays a prominent role in the prokaryotic SOS response, in mammalian cells so far no enhenaced as a result of treatment with DNA-damaging agents of the cells, rather than of infecting viruses, has been found. In order to study recombination as a UV-inducible cellular phenomenon we infected UV-irradiated normal and repair-deficient human fibroblasts with a mixed population of adenovirus 5 (Ad5) mutants that carried a deletion in the E1A of the E2A gene. Wild-type recombinant progeny viruses were readily obtained, but no enhanced recombination was observed at any UV dose given to the cells, nor at any time point between −6 h and +4 days between irradiation and infection. Control experiments, in which we infected unirradiated cells with UV-irradiated Ad5 deletion mutants (a test for recombination targeted at UV-damaged DNA) showed a strong increase in wild-type recombinant viruses when both deletion mutants had been irradiated compared to the additive effect of irradiation of either one of the mutants alone. Therefore, this study shows that UV irradiation enhanced recombination activity in cells that is specially targeted to damaged DNA, but it does not cause a general (untargeted) recombinational response (enhanced recombination) in the cell.     

Alu-mediated nonallelic homologous and nonhomologous recombination in the BMPR2 gene in heritable pulmonary arterial hypertension

PurposeThe purpose of this study was to undertake thorough genetic analysis of the bone morphogenetic protein type 2 receptor (BMPR2) gene in patients with pulmonary arterial hypertension.MethodsWe conducted a systematic analysis for larger gene rearrangements together with conventional mutation analysis in 152 pulmonary arterial hypertension patients including 43 patients diagnosed as having idiopathic pulmonary arterial hypertension and 10 diagnosed as having familial pulmonary arterial hypertension.ResultsAnalysis of the BMPR2 gene revealed each of the four kinds of nonsense and frameshift mutations, one missense mutation, one splice-site mutation, and two types of exonic deletion. For cases in which exons 1–3 were deleted, the 5′ and 3′ break points were located in the AluY repeat sequences in the 5′ side of the adjacent NOP58 gene and in the AluY repeat sequences in intron 3, suggesting an AluY-mediated nonallelic homologous recombination as the mechanism responsible for the deletion. For the case in which exon 10 was deleted, nonhomologous recombination took place between the AluSx site in intron 9 and a unique sequence in intron 10.ConclusionExonic deletions of BMPR2 account for at least part of BMPR2 mutations associated with heritable pulmonary arterial hypertension in Japan, as previously reported in other populations. One of our cases was mediated via Alu-mediated nonallelic homologous recombination and another was mediated via nonhomologous recombination.Genet Med15 12, 941–947.     

Common fragile site expression and genetic predisposition to breast cancer

The expression of common fragile sites induced by aphidicolin and caffeine was evaluated on prometaphase obtained from the peripheral blood lymphocytes of 35 women with breast cancer, their 35 clinically healthy female family members, and 20 sex- and age-matched normal controls. As a result of the cytogenetic and statistical evaluation, the number of damaged cells, chromosomal aberrations, and expression frequencies of fragile sites detected in patients with breast cancer and their first-degree relatives were found to be significantly higher than those in the control group. Our findings indicate an increased genetic instability in women with breast carcinomas and their relatives. Therefore, fragile sites may be used as a reliable marker for defining genetic susceptibility to cancer in general.     

Current advances in DNA repair: regulation of enzymes and pathways involved in maintaining genomic stability

Novel discoveries in the DNA repair field have lead to continuous and rapid advancement of our understanding of not only DNA repair but also DNA replication and recombination. Research in the field transcends numerous areas of biology, biochemistry, physiology, and medicine, making significant connections across these broad areas of study. From early studies conducted in bacterial systems to current analyses in eukaryotic systems and human disease, the innovative research into the mechanisms of repair machines and the consequences of ineffective DNA repair has impacted a wide scientific community. This Forum contains a select mix of primary research articles in addition to a number of timely reviews covering a subset of DNA repair pathways where recent advances and novel discoveries are improving our understanding of DNA repair, its regulation, and implications to human disease.     

Architectural flexibility in lambda site-specific recombination: three alternate conformations channel the attL site into three distinct pathways

Background :  In the phage lambda life cycle, the Integrase (Int) protein carries out recombination between two different sets of DNA substrates: attP and attB in integration, attL and attR in excision. In each case, the partners are very different in structure from each other and the recombination reaction between them is effectively irreversible. For comparison, we have studied the recombination mediated by Int between two identical attL sites. Both in vitro and in vivo , recombination between two attL sites can be mediated inefficiently by Int alone. But, while IHF can stimulate recombination 5–10-fold in vivo (to the level of excision and integration), this stimulation is not observed under standard conditions in vitro
Results :  We find that IHF can stimulate the in vitro recombination between two attL s that are modified to be defective in one of the high affinity binding sites for Int, P'1. With such substrates, the efficiency of IHF-stimulated recombination is comparable to that seen in vivo . The requirements for this reaction distinguish it from other lambda recombination pathways, as does the performance of several mutant Int proteins. Recombination of attL sites on intracellular plasmids suggests that this pathway is effective in vivo , but that some unknown factor or condition permits it to operate on wild-type as well as mutated attL sites.
Conclusions :  The recombination pathway described in this work apparently uses a unique attL architecture, one which requires bending by IHF and is inhibited by Int bound at the P'1 site. In addition to demonstrating the architectural flexibity of the lambda system, this pathway should be a valuable resource for separating the basic requirements of strand exchange chemistry from the features which impart directionality.     

Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain

Genetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics. Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes. Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages. In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L. lactis host. They occurred by homologous recombination with phage-related sequences present in the host chromosome. Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication. We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages.