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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. Homologous recombination (HR)1 is an essential process in mammalian cells for the reassortment of genetic material and for the repair of DNA damage. RAD51 is the key eukaryotic protein for HR, mediating homologous pairing of DNA sequences and strand exchange (1). Five novel RAD51-like proteins have recently been identified in mammalian cells; these proteins have limited homology to RAD51, and their functions are relatively unknown (2, 3). The RAD51-like protein RAD51L2 (RAD51C) was first identified from data base searches through partial homology to RAD51 and other members of this protein family (4). Recently, we (5) and others (6) have described DNA damage-sensitive cell lines that specifically lack RAD51L2 activity. It was shown that the RAD51L2 gene function was not redundant to other RAD51-like genes or RAD51 itself, because only RAD51L2 was able to restore DNA damage resistance to these cell lines. It was further suggested that RAD51L2 has a role in RAD51-dependent HR because RAD51L2-deficient cells show a reduction in sister-chromatid exchange and a decrease in damage-dependent RAD51 focus formation (5, 6). However, a direct role in homologous recombination processes has not been shown.Human RAD51L2 shares only 27% sequence identity with the human RAD51 protein, with the most well conserved residues being those with homology to the nucleotide-binding or "Walker motif " (2). The Walker motif (7) consists of two separate sequences, A and B, which come together to form a nucleotide binding site. These residues are highly conserved in the RAD51 family and in its prokaryotic counterpart RecA. The Walker A motif, also known as the phosphate-binding loop (P-loop), is defined by the consensus sequence (G/AXXXXGKT/ S), w...

Genes from the recA/RAD51 family play essential roles in homologous recombination in all organisms. Using sequence homologies from eukaryotic members of this family we have identified fragments of two additional mammalian genes with homology to RAD51. Cloning the full-length cDNAs for both human and mouse genes showed that the sequences are highly conserved, and that the predicted proteins have characteristic features of this gene family. One of the novel genes (R51H2) occurs in two forms in human cDNA, differing extensively at the 3' end, probably due to an unusual form of alternative splicing. The new genes (R51H2 and R51H3) were mapped to human chromosomes 14q23-24 and 17q1.2, respectively. Expression studies showed that R51H2 is expressed at lower levels than R51H3 , but that expression of both genes occurs at elevated levels in the testis compared with other tissues. The combination of gene structure conservation and the transcript expression patterns suggests that these new members of the recA/RAD51 family may also function in homologous recombination-repair pathways.

In an attempt to identify trans-acting factors involved in replication origin function, we have characterized the RAR3 and RAR5 genes, identified by mutations which increase the mitotic stability of artificial chromosomes whose replication is dependent on the activity of weak ARS elements. Sequence analysis has shown that the RAR3 gene is identical to GAL11/SPT13, which encodes a putative transcription factor involved in the expression of a wide range of genes. Change-of-function mutations that truncate the RAR3 protein appear to be required to enhance chromosome stability. In contrast, loss of the RAR5 protein results in enhanced chromosome stability, as if the protein is an inhibitor of ARS function. The RAR5 gene encodes the 175 kDa DNA strand transfer protein beta, an activity that can promote the transfer of a strand from a double-stranded DNA molecule to a complementary single strand. This observation implies that a presumed recombination activity can affect eukaryotic chromosomal replication.

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