Mutagenesis of the P-loop Motif in the ATP Binding Site of the RecA Protein from Escherichia coli

100

103

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK a of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.

Section: Methodsmentioning

confidence: 99%

C-terminal Deletions of the Escherichia coli RecA Protein

Lusetti

Wood

Fleming

et al. 2003

100

103

“…The Walker A motif, also known as the phosphate-binding loop (P-loop), is defined by the consensus sequence (G/AXXXXGKT/ S), where X is any amino acid. In RecA, non-conservative mutation of any of the four residues that identify the P-loop results in a non-functional protein but restricted mutation at other positions is tolerated (8,9). A well characterized mutation in this motif is the conservative lysine to arginine (K72R) substitution: this mutant protein is able to bind but not hydrolyze ATP and can promote limited strand exchange in vitro (10).…”

Section: Homologous Recombination (Hr)mentioning

confidence: 99%

Identification of Functional Domains in the RAD51L2 (RAD51C) Protein and Its Requirement for Gene Conversion

French¹,

Tambini

Thacker

2003

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...

“…Consistent with this degree of conservation, the mutation of this residue in ATPbinding proteins generally compromises function. This is certainly true for Escherichia coli RecA for which even the relatively conservative K72R mutation gives a null phenotype (32) although the mutant protein retains the ability to promote homologous pairing of DNA strands (33). S. cerevisiae Rad51p altered at this P-loop lysine similarly has a compromised function; the K191A mutation gives a null phenotype, whereas K191R has a partially defective phenotype (34).…”

Section: Site-directed Mutations Of the Walker Box A Of Xrcc2 Have LImentioning

confidence: 99%

XRCC2 Is a Nuclear RAD51-like Protein Required for Damage-dependent RAD51 Focus Formation without the Need for ATP Binding

O'Regan¹,

Wilson

Townsend

et al. 2001

The human XRCC2 gene was recently identified by its ability to complement a hamster cell line, irs1, which is sensitive to DNA-damaging agents and shows genetic instability. The XRCC2 protein is highly conserved in mammalian species and has structural features, including a putative ATP-binding domain (P-loop), consistent with membership of the RecA/RAD51 family of recombination-repair proteins. We show that a hybrid XRCC2-green fluorescent protein, which was found to be functional by complementation, localizes to the nucleus. We have established a functional link between XRCC2 and RAD51 by looking at damage-dependent RAD51 focus formation in the irs1 cell line. Little or no formation of RAD51 foci occurred in irs1. This effect was specific to the loss of XRCC2 because transfection of the gene into irs1 restored normal levels of focus formation. Surprisingly, XRCC2 genes carrying site-specific mutations in P-loop residues were found to be able to complement the XRCC2-deficient irs1 line for a number of different end points. We conclude that XRCC2 is important in the early stages of homologous recombination in mammalian cells to facilitate RAD51-dependent recombination repair but that it does not make use of ATP binding to promote this function.The repair of DNA damage by homologous recombination has an important function in maintaining genetic stability in cells. In bacteria, the RecA protein has a central role in the recombination process, and in the last decade RecA-like proteins have been discovered in eukaryotes. In particular the RAD51 protein is highly conserved from yeast to humans and has been shown to have similar attributes to RecA (1, 2). Mutations in both RecA and RAD51 cause severe defects in recombination and extreme sensitivity to DNA-damaging agents. RecA acts directly in recombination processes in which, in the presence of ATP, it forms a polymer on single-stranded DNA and promotes strand exchange with a homologous sequence (3). Using molecular recombination assays, yeast (Saccharomyces cerevisiae) and human RAD51 proteins have been shown to promote strand exchange similarly, although some of the biochemical properties of RAD51 differ from those of RecA.For example, purified RecA preferentially binds to singlestranded DNA and hydrolyzes ATP at a relatively high rate, whereas the yeast and human RAD51 proteins bind equally to single-and double-stranded DNA and show a much lower rate of ATP hydrolysis (1). All members of this family have a highly conserved sequence motif, first described by Walker et al. (4), that has been linked to ATP binding. The flexible loop of this motif (Walker box A) interacts with the phosphates of ATP and is therefore sometimes called the P-loop.In S. cerevisiae, two further members of the RecA/ RAD51family of proteins facilitate homologous recombination in mitotic cells; Rad55p and Rad57p form a heterodimer and stimulate RAD51-mediated recombination (5). Yeast mutants that lack these recombination proteins are also extremely sensitive to agents causing severe forms of da...