ABSTRACT
Rad51C is a central component of two complexes formed by five Rad51 paralogs in vertebrates. These complexes are involved in repairing DNA double-strand breaks through homologous recombination. Despite accumulating evidence suggesting that the paralogs may prevent aneuploidy by controlling centrosome integrity, Rad51C's role in maintaining chromosome stability remains unclear. Here we demonstrate that Rad51C deficiency leads to both centrosome aberrations in an ATR-Chk1-dependent manner and increased aneuploidy in human cells. While it was reported that Rad51C deficiency did not cause centrosome aberrations in interphase in hamster cells, such aberrations were observed in interphase in HCT116 cells with Rad51C dysfunction. Caffeine treatment and down-regulation of ATR, but not that of ATM, reduced the frequency of centrosome aberrations in the mutant cells. Silencing of Rad51C by RNA interference in HT1080 cells resulted in similar aberrations. Treatment with a Chk1 inhibitor and silencing of Chk1 also reduced the frequency in HCT116 mutants. Accumulation of Chk1 at the centrosome and nuclear foci of gamma H2AX were increased in the mutants. Moreover, the mutant cells had a higher frequency of aneuploidy. These findings indicate that the ATR-Chk1 pathway plays a role in increased centrosome aberrations induced by Rad51C dysfunction.
Subject(s)
Cell Cycle Proteins/metabolism , Centrosome/ultrastructure , DNA-Binding Proteins/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Aneuploidy , Ataxia Telangiectasia Mutated Proteins , Cell Line, Tumor , Checkpoint Kinase 1 , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Humans , RNA Interference , Recombination, GeneticABSTRACT
XRCC3 was inactivated in human cells by gene targeting. Consistent with its role in homologous recombination, XRCC3(-/-) cells showed a two-fold sensitivity to DNA cross-linking agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation and elevated chromosome aberrations. Furthermore, endoreduplication was increased five- seven-fold in the mutants. The T241M variant of XRCC3 has been associated with an increased cancer risk. Expression of the wild-type cDNA restored this phenotype, while expression of the variant restored the defective recombinational repair, but not the increased endoreduplication. RPA, a protein essential for homologous recombination and DNA replication, is associated with XRCC3 and Rad52. Overexpression of RPA promoted endoreduplication, which was partially complemented by overexpression of the wild-type XRCC3 protein, but not by overexpression of the variant protein. Overexpression of Rad52 prevented endoreduplication in RPA-overexpressing cells, in XRCC3(-/-) cells and in the variant-expressing cells, suggesting that deregulated RPA was responsible for the increased endoreduplication. These observations offer the first genetic evidence for the association between homologous recombination and replication initiation having a role in cancer susceptibility.
Subject(s)
Agmatine/analogs & derivatives , DNA Repair/physiology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/metabolism , Ploidies , Recombination, Genetic , Agmatine/pharmacology , Cell Line , Chromosomes, Human/genetics , Chromosomes, Human/metabolism , Cross-Linking Reagents/pharmacology , DNA Damage/drug effects , DNA Repair/genetics , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , Gene Deletion , Gene Expression , Gene Targeting , Humans , Mitomycin/pharmacology , Rad52 DNA Repair and Recombination Protein , Replication Protein A , Succinates/pharmacologyABSTRACT
Complementary DNA (cDNA) arrays were used to detect highly expressed messenger RNA (mRNA) at postnatal day 2 (P2) and P10 in the mouse eye, and several clones highly expressed at P2 were isolated. We focused among them on a novel gene, the ocular development-associated gene (ODAG), which was down regulated at P10. The expression around birth was subsequently confirmed by reverse transcription-polymerase chain reaction. Mouse ODAG cDNA encodes a protein of 266 amino acids. Human ODAG cDNA and genomic structure were identified by basic local alignment search tool analysis of the GenBank database with mouse ODAG. Mouse ODAG-specific mRNA expression was detected in various mouse tissues within the eye at P2 and P7, whereas it was not detected anywhere at P14, suggesting that ODAG may play a role in eye development.
Subject(s)
Eye Proteins/genetics , Eye/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/chemistry , DNA, Complementary/genetics , Eye/growth & development , Female , Gene Expression Regulation, Developmental , In Situ Hybridization , Male , Mice , Mice, Inbred C3H , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.
