The Central Aromatic Residue in Loop L2 of RecA Interacts with DNA

Abstract

DNA repair is vital for both prokaryotic and eukaryotic cells. RecA is an ubiquitous and multi-functional enzyme involved in various steps of DNA repair: regulation of synthesis of DNA repair proteins (SOS induction), promotion of homologous recombination, and mutagenesis (For reviews, see Refs. 1-3). For these activities, RecA first binds to single-stranded DNA with a strong cooperativity and forms a filamentous complex in which RecA subunits are arranged in a helical manner around the DNA (4). This nucleofilament binds a second doublestranded DNA molecule for the strand exchange reaction (5) and interacts with repressors for the induction of the SOS system (6) and with UmuD and UmuDЈ proteins for mutagenesis (7,8).The molecular structure of the RecA filament in the absence of DNA has been determined by x-ray crystallography (9). A lower resolution structure of the nucleofilament (4) and of the DNA in the complex (5) has also been determined. These studies have shown that the DNA strands lie near the axis of the RecA-DNA filament. Determination of the DNA binding sites in RecA would allow us to build a higher resolution model of the RecA-DNA complex. Despite various studies, however, the DNA binding site(s) of RecA has not yet been determined. From the comparison of sequence with the DNA binding domain of ssDNA 1 binding protein of the Ike phage, GP5 protein, the domain comprising residues 240 -310 has been proposed as a DNA binding site (10 -12). But this region is in the subunitsubunit interface in the crystal of the RecA filament (9). Story et al. (9) rejected this possibility and proposed that two disordered loops that could not be traced in the x-ray analysis were the DNA binding domains because these loops line the cavity running down the middle of the RecA filament. In fact, mutations in L2 loop affect the activities of RecA (13), although modifications in L1 loop are easily tolerated (14).Limited proteolysis of RecA-DNA complexes in the presence of ATP␥S has shown that a peptide spanning loop L2 is protected from tryptic digestion (15). In addition, peptides derived from this region, including a 20-amino acid peptide from residues 193-212 can bind to ssDNA with a high affinity. Subsequent work has shown that such a peptide can also bind to dsDNA. In addition, a phenylalanine at position 203 in loop L2 that is highly conserved between prokaryotic RecAs and their eukaryotic homologues (16, 17) plays a central role in the binding. Only aromatic acid substitutions allow the peptides to bind to both ss-and dsDNA. 2 With the purpose of examining the possibility that this central aromatic residue in loop L2 interacts with DNA, we inserted a tryptophan residue in this loop in the place of phenylalanine. An interaction with DNA could then be detected by a change in the tryptophan fluorescence. Our results show that the modified protein is active, and its fluorescence is strongly decreased upon binding to DNA.

Design and Evaluation of a Tryptophanless RecA Protein with Wild Type Activity

“…To demonstrate the utility of the W-less RecA protein as a "background" for subsequent Trp substitutions, we constructed and purified F203W and F203W-W290H-W308F mutant RecA proteins. The Trp for Phe203 substitution was shown previously to provide a reporter for RecA-DNA interactions (12). As shown by the relative quench of emission in Figs.…”

“…For example, Trp replacement of His163 in loop 1 of the RecA protein allowed the formation and conformational dynamics of RecA-ATP␥S filaments to be monitored by real-time spectrofluorometry (15)(16)(17). In addition, mutant proteins such as F203W (12) and Y65W (18) report DNA binding, while Y264W and Y103W (19,20) report binding ATP as well as DNA.…”

“…Unfortunately the natural DNA bases are minimally luminescent (7) and the wild type RecA tryptophans do not serve as reporters for binding of DNA (8 -11) or ATP (12). Moreover, the use of extrinsic fluorophores such as fluorescein or ANS is not optimal because of the potential of the reporter moiety to influence the interactions under study (13,14).…”

A RecA / RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only

“…This is the first demonstration of complementation of E. coli recA defect by a non-bacterial RecA homologue. Pk-REC lacks the flexible L2 loop region which promotes RecA binding to ss and dsDNA (Maraboeuf et al 1995). Pk-REC and eukaryotic RecA homologues (RAD51 and DMC1) possibly utilize different binding motifs.…”

DNA recognition of a 24-mer peptide derived from RecA protein

“…21,[23][24][25][26][27][28][29] We also have published quantitative data of the interactions between peptides, 30 -33 and new quantitative biotechnologies in combinatorial chemistry. 34,35 These information is also very useful for the design of regulation materials for nucleic acid and peptide functions.…”