Identification of functional domains in the RAD51L2 (RAD51C) protein and its requirement for gene conversion.
The RAD51 protein plays a key part in the process of homologous recombination through its catalysis of homologous DNA pairing and strand exchange. Additionally five novel mammalian RAD51-like proteins have been identified in mammalian cells, but their roles in homologous recombination are much less well established. These RAD51-like proteins form two different complexes, but only the RAD51L2 (RAD51C) protein is a part of both complexes. By using site-directed mutagenesis of RAD51L2, we show that non-conservative mutation of the putative ATP-binding domain severely reduces its function, whereas a conservative mutation shows partial loss of function. We find that the protein is localized to the nucleus by tagging RAD51L2 with the green fluorescent protein and provisionally identify a C-terminal domain that acts as a nuclear localization signal. Further, a RAD51L2-deficient cell line was found to have significantly reduced homology-directed repair of a DNA double-strand break by gene conversion. This recombination defect could be partially restored by ectopic expression of the human RAD51L2 protein. Therefore we have identified protein domains that are important for the correct functioning of RAD51L2 and have shown that there is a specific requirement for RAD51L2 in gene conversion in mammalian cells.
French
Catherine A
CA
Medical Research Council, Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD, England.
Tambini
Cathryn E
CE
Thacker
John
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005785:Gene Conversion; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C448305:RAD51C protein, human; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate