A genetic analysis of RecA-LexA protein interactions in Escherichia coli

摘要: The RecA protein of Escherichia co/ils a small involved in many important functions including homologous recombination, mutagenic repair, regulation the SOS system, and prophage induction. In normally growing cells, is an inactive form. However, when cells are subjected to DNA damage, forms helical filament with single stranded ATP. This ternary complex activated form RecA. A key step several these processes mediated cleavage different proteins. not classic protease, but instead causes proteins undergo autodigestion. main goal this research was investigate role that plays by determining what residues interact substrates LexA, UmuD X CI. possible model for binding cleft formed between two adjacent monomers has been proposed. Site-directed mutagenesis 14 used change map cleft. An analysis previously characterized recA mutants also suggested other regions might substrates. residues. Candidate were changed alanine order reduce side chain contacts while minimizing perturbation folding. mutant then ability do recombination ONA examined mediate Several showed some defects LexA or X. CI, being proficient functions. fact selectively defective one protein, others, suggests they can filaments. Most mutations differentially affected CI mapped outside region. result bind cleft, cleavable may groove turns filament. 15 Chapter 1: Introduction 1.1 Biological Functions coli multifunctional damage inducible genes. Cells function sensitive unable after conjugal mating, have chromosomal segregation. well conserved prokaryotes, over sixty eubacterial genes isolated from organisms gram positive bacteria, proteobacteria, mycoplasma, cyanobacteria (Karlin Brocchieri, 1996). Homologous similar eukaryotes flys, yeast, humans (Brendel et al., 1997)(McKee 1996)(Li 1997). monomer relatively 352 amino acids; however all its activities require formation polymeric vitro, (ssDNA) ATP, forming (RecA*) along DNA. vivo, sources ssDNA arise action 16 RecBCD enzyme, replisomes stalled at sites create gapped To carry out process must separate molecules initial molecule on which forms. repair damaged using RecA* now second mechanism searches homology secondary double (dsDNA) unknown, three promoted as intermediate (Kurumizaka Shibata, 1996)(Mazin Kowalczykowski, 1996)(Voloshin Camerini-Otero, As playing direct via indirect regulating expression presence triggers global cellular response known response. repressor regulates group more than 20 called operon (Lewis 1994). These include recA, lex umuDC, whose 17 products repair. repressor, since regulated leads higher levels cell. (See figure 1.1) repaired, drop no longer cleaved. allows increase once again repress It interesting note addition highly conserved, system appears be present bacteria. £ coH promoter determined repressed during normal growth, induced negative genera (Fernandez de Henestrosa 1991), implying inactivation repressor. lexA sequenced (Garriga 1992)(Riera Also, R subtilus homolog, DinR, cleaved E RecA, B. cleaves (Winterling promotes UmuD, contrast inactivated cleavage, product, UmuD', active protein. UmuD' UmuC Involved