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Replication fork reversal in DNA polymerase III mutants of Escherichia coli : a role for the β clamp
Author(s) -
Grompone Gianfranco,
Seigneur Marie,
Ehrlich S. Dusko,
Michel Bénédicte
Publication year - 2002
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2002.02962.x
Subject(s) - biology , recbcd , dna polymerase , dna replication , processivity , microbiology and biotechnology , mutant , dna polymerase i , dna clamp , replication factor c , polymerase , holliday junction , dna polymerase ii , genetics , dna , control of chromosome duplication , dna repair , gene , polymerase chain reaction , reverse transcriptase
Summary Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four‐way junction. RuvABC‐catalysed resolution of this Holliday junction causes chromosome double‐strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC‐dependent DSBs caused by the dnaEts mutation occurred at 37°C or 42°C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC‐dependent DSB caused by the dnaNts mutation occurred only at semi‐permissive temperature, 37°C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42°C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.