Functionally important residues at a subunit interface site in the RecA protein from Escherichia coli.

M.C Skiba,K.L Knight

doi:10.1016/s0021-9258(17)41934-x

Abstract

Assembly of RecA subunits into long, helical oligomers is required for its roles in recombinational DNA repair and homologous genetic recombination. The crystal structure of RecA reveals an extensive network of amino acid residues that lie at the subunit boundaries. We have introduced a large set of substitutions at 5 clustered residues, which are shown in the crystal structure to make specific contacts with positions in the neighboring monomer. We find that 3 of the 5 residues are important for RecA function (Lys216, Phe217, and Arg222), whereas the other 2 (Asn213 and Tyr218) are not. The patterns of functionally allowed substitutions provide insight into the chemical and steric constraints required at these positions.

Highlights

Of Redsubunits into long, helical oligomemrsakes specific contacts with the neighboring subunit and, in is required for ita roles in recombinationaDl NA repair some cases, with positionsin the same subunit
1) Which of the predicted intersubunit, as introduced a large set of substitutions at 5 clustered well as intrasubunit, interactions are important for residues, which are shown in the crystal structure to RecA function, i.e. which positions tolerate only very conservmake specific contacts with positions in the neighboartiivnegor no substitutions?2) What canbe determined about the monomer
Analysis of the R e d Crystal Structure-Residues predicted to parpositions

Summary

MATERIALS AND METHODS

Analysis of the R e d Crystal Structure-Residues predicted to parpositions. ticipate in monomer-monomer interactions are defined as those for which solvent-accessible surface area decreases bymore than 15 Az upon oligomer formation (6).Fifty-five of the 303 amino acids in the. 5)For locations of those amino acidresidues whose solvent accessible the guanidinium group is positioned such that it can form an surface area decreases dramatically upon polymer formation ionic interaction with the side chain of Glum within the same subunit, suggest that six areas of the protein structure play critical roleass well as H-bonding interactions with the side chain of Hiss[7] on the in subunit interactions (6).This study focuses on oneof these areas, which is located at residues 213-222 (Fig. L4).Analysis of the RecA crystal structure shows that 5 amino acid side chains in this region (Asn213,Lys216,Phe2I7,TyPs, andAr 8 2 2 ) are directed outward towardthe neighboring subunit and each neighboringsubunit. None of the mutanta in this study showeda steady state level of RecA protein significantlydifferent from that of wild type RecA (data not shown)

RESULTS AND DISCUSSION

TABLEI Activity of RecA protein mutants in vivo

Ala np