XthA (Exonuclease III) regulates loading of RecA onto DNA substrates in log phase Escherichia coli cells.
Exonucleases can modify DNA substrates created during DNA replication, recombination and repair. In Escherichia coli, the effects of several 3'-5' exonucleases on RecA loading were studied by assaying RecA-GFP foci formation. Mutations in xthA (ExoIII), xseAB (ExoVII), xni (ExoIX), exoX (ExoX) and tatD (ExoXI) increased the number of RecA-GFP foci twofold to threefold in a population of log phase cells grown in minimal medium. These increases depend on xonA. Epistasis analysis shows that ExoVII, ExoX, ExoIX and ExoXI function in a common pathway, distinct from ExoIII (and ExoI is upstream of both pathways). It is shown (paradoxically) that in xthA mutants, RecA-GFP loading is predominantly RecBCD-dependent and that xthA recB double mutants are viable. Experiments show that while log phase xthA cells have twofold more double-stranded breaks (DSBs) than wild type, they do not induce the SOS response. The increase in RecA loading is independent of the base excision repair (BER) proteins Nth, MutM and Nei. It is proposed that log phase cells produce DSBs that do not induce the SOS response. Furthermore, ExoI, ExoIII and the other 3'-5' exonucleases process these DSBs, antagonizing the RecBCD pathway of RecA loading, thus regulating the availability of these substrates for recombination.
Molecular and Cellular Biology Graduate Program, Morrill Science Center, University of Massachusetts at Amherst, Amherst, MA 01003, USA.
Richard C
RC
Centore
Roxane
R
Lestini
Steven J
SJ
Sandler
D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004843:Epistasis, Genetic; D048168:Escherichia coli K12 / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050296:Microbial Viability; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013014:SOS Response, Genetics
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III