recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities.
Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination.
Liu
S K
SK
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Eisen
J A
JA
Hanawalt
P C
PC
Tessman
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009038:Motion; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship
D017931:DNA Primers; D046911:Macromolecular Substances; D016685:Mitomycin; D011938:Rec A Recombinases; D010450:Endopeptidases