Roles of PriA Protein and Double-Strand DNA Break Repair Functions in UV-Induced Restriction Alleviation inEscherichia coli

Abstract

It has been widely considered that DNA modification protects the chromosome of bacteria E. coli K-12 against their own restriction-modification systems. Chromosomal DNA is protected from degradation by methylation of target sequences. However, when unmethylated target sequences are generated in the host chromosome, the endonuclease activity of the EcoKI restriction-modification enzyme is inactivated by the ClpXP protease and DNA is protected. This process is known as restriction alleviation (RA) and it can be induced by UV irradiation (UV-induced RA). It has been proposed that chromosomal unmethylated target sequences, a signal for the cell to protect its own DNA, can be generated by homologous recombination during the repair of damaged DNA. In this study, we wanted to further investigate the genetic requirements for recombination proteins involved in the generation of unmethylated target sequences. For this purpose, we monitored the alleviation of EcoKI restriction by measuring the survival of unmodified l in UV-irradiated cells. Our genetic analysis showed that UV-induced RA is dependent on the excision repair protein UvrA, the RecA-loading activity of the RecBCD enzyme, and the primosome assembly activity of the PriA helicase and is partially dependent on RecFOR proteins. On the basis of our results, we propose that unmethylated target sequences are generated at the D-loop by the strand exchange of two hemi-methylated duplex DNAs and subsequent initiation of DNA replication.

Mechanistic Analysis of a DNA End Processing Pathway Mediated by the Xenopus Werner Syndrome Protein

“…Although none of the RecQ helicases in other organisms is essential for homologous recombination and, by inference, for ds-DNA end processing, the coupled end unwinding/ss-DNA degradation might be carried out by multiple combinations of helicases and ss-DNA nucleases. Indeed, the helicases and the nucleases of the two E. coli end processing pathways, RecBCD and RecQ/RecJ, are known to be able to act in various combinations (41). In higher eukaryotes, there are multiple RecQ helicases.…”

Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival

“…Alleviation of this defense system is thought to occur when DNA damage activates mechanisms that produce unmethylated target sequences, usually generated by homologous recombination, within the bacterial chromosome (44). Interestingly, sfiA priA strains are reduced in their ability to activate UV-induced restriction alleviation, but the activity is almost completely restored by either the priA300 or dnaC809 mutations (24). The requirement for PriA in UV-induced restriction alleviation may reflect loading of DnaB onto structures formed by recombination.…”

RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA

“…2B). Under slightly different conditions, with a longer DNA substrate and in the presence of 5% PEG10K, the ATPase activity of the RecFR complex was lower than that of RecF alone in the presence of dsDNA (supplemental Table S1, lane ds (30)) (35). The data suggest that RecR may stabilize interaction between RecF and dsDNA via two mechanisms: first, by increasing the affinity of RecF to dsDNA, and, second, by decreasing the ATPase rate of RecF in the presence of dsDNA.…”