Identification oftheuvrDGeneProduct ofSalmonella typhimurium LT2

Theproduct oftheuvrDgeneofSalmonella typhimurium LT2andEscherichia coli K-12isthought toplay arole inboththecorrection ofmismatched basesand therepair ofDNA damage, since insertion mutations intheuvrDgeneincrease the spontaneous mutation frequency andmakethecells more sensitive tokilling by UV irradiation. Toclone theuvrDgeneofS. typhimurium, we first generated a uvrD-specific probe byusing DNA fromanS.typhimurium uvrD421::TnS mutant. Thisprobe was usedtoscreena library ofS. typhimurium DNA.Bacteriophage carrying intact uvrD+geneswere subsequently identified, andtheuvrD+gene was subcloned ontoalow-copy-number vector. Byusing acombination ofTnlOOO insertion mutagenesis andthemaxicell technique, theproduct oftheuvrDgene was showntobea75,000-dalton protein, andtherelative direction oftranscription ofthisprotein was determined. Introduction ofa low-copy-number plasmid carrying theS.typhimurium uvrD+geneinto uvrDinsertion mutantsofeither S. typhimurium orE.coli restored thespontaneous mutation frequency anddegree ofUV sensitivity tothelevels inthecorresponding uvrD+strains. Recent studies havesuggested that animportant system forthemaintenance ofgenetic fidelityinanumberofprocaryotes maybeamismatchrepair system. Ithasbeenpostulated that this system usesmethylation attheN6position ofadenine inGATCsequences todiscriminate daughter strands fromparental strands atthe replication fork(5,14,18,19,33,36,37). The products ofthespontaneous mutatorlociin Escherichia coli, mutH,mutL,mutS, anduvrD, appear toplayroles inthismethyl-instructed mismatch repair system (5, 6,18,19). We havepreviously reported theisolation of mutH,mutL,mutS, anduvrDmutants ofSalmonella typhimurium LT2that weregenerated by insertion oftransposable elements TnSandTnJO (26). We areinterested instudying themechanismofthis mismatch repair system andaspart ofthis study wished toclone these genes. Todo this, we decided tousea strategy thathas proved useful incloning oftheumuClocus ofE. coli (S.J.Elledge andG.C.Walker, J.Mol. Biol., inpress) namely, thestrategy ofusing insertion mutants asawayofgenerating probes tothegeneofinterest. Thefirst genewhichwe undertook toclone wastheuvrDgeneofS. typhimurium. TheuvrDgeneproduct mayplaydirect or