DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide

Abstract

Escherichia coli possesses three DNA polyrnerases (17). DNA polymerase I has been implicated in DNA repair, since mutations at the polA locus increase sensitivity to many DNA-damaging agents, including UV irradiation (7), methyl methanesulfonate (MMS) (10), hydrogen peroxide (1), and bleomycin (25). No role has yet been discovered for DNA polymerase II. DNA polymerase III has a central role in DNA replication. Temperature-sensitive (ts) mutations of the dnaE (polQ gene coding for the a-subunit of DNA polymerase III are lethal at 43°C, the restrictive temperature (11,30). Cells lacking the a-subunit function of the holoenzyme are inviable, regardless of whether DNA polymerase I or II is present. Studies on the role of DNA polymerase III in DNA repair have been limited due to the inviability of the mutants at the restrictive temperature, but have implicated DNA polymerase III in excision and post-replication repair of UV damage (2,9,16,25,26,29), in repair of damage due to X rays (3,14,28), and possibly in recombination and mutagenesis (2-4, 13).The pcbAl genotype has been shown to suppress phenotypically three different polC(Ts) mutations if a functional DNA polymerase I is present (5,22,23 show an increase in sensitivity at 430C, where DNA polymerase III is inactive (5). This is compatible with our earlier observations suggesting a role in DNA repair for DNA polymerase III (9) and has led us to the current studies. In this paper, we report the results of testing these mutants for sensitivity to several different DNA-damaging agents. By quantitative cell survival as well as alkaline sucrose sedimentation, we show that DNA polymerase III is necessary for the optimal repair of MMS-and hydrogen peroxide-* Corresponding author.induced DNA damage but is not strictly required for the repair of damage by UV light, bleomycin, or psoralen. MATERIALS AND METHODS

Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli

“…These strains were significantly more sensitive to hydrogen peroxide at the restrictive temperature at which DNA polymerase III is inactive than at the permissive temperature (7). It is worth noting that the level of DNA polymerase I, whether normal or partial, does not affect the results of this comparison (7). These strains were also more hydrogen peroxide sensitive than MH91, which carries only pcbAl (Table 2).…”

“…This X strain was provided by G. Weinstock and contains the recA promoter fused to the lacZ gene (13 tightly linked to gyrB. These strains were significantly more sensitive to hydrogen peroxide at the restrictive temperature at which DNA polymerase III is inactive than at the permissive temperature (7). It is worth noting that the level of DNA polymerase I, whether normal or partial, does not affect the results of this comparison (7).…”

“…The presence of the recA mutation was verified by sensitivity to UV irradiation. MH91 contains the pcbAl mutation in a wild-type background (7). The strain W3L is a X lysogen with XGE190.…”

“…In addition, various mutants of Escherichia coli have been shown to be sensitive to this agent. These mutants include those deficient in RecA protein (recA) (1, 3), exonuclease III (xthA) (5), DNA polymerase I (polA) (1,3,8), and DNA polymerase III (polC) (7). The interaction of these enzymes in the repair of hydrogen peroxide damage has not been determined.…”

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46

“…At various intervals, duplicate or triplicate aliquots were removed, quickly diluted in M9 salts, plated, and incubated at 32°C to determine survivors. MMS and bleomycin were used at final concentrations of 38 mM and 10 ,ug/ml, respectively, according to Hagensee et al (26).…”