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PriA and RecBCD functions cooperate to create a signal for UV-induced RA in Escherichia coli

It has been widely considered that DNA modification is a defense mechanism of bacteria against their own restriction-modification systems. By methylating specific adenine or cytosine residue within target nucleotide sequences, restriction endonulcease protect their DNA from degradation. But, under certain conditions, for example after UV irradiation of E. coli K-12 bacteria, unmodified target sequences for EcoKI enzyme are temporarily generated in the chromosome. Such target sequences are not cleaved by EcoK restriction system since the activity of EcoKI restriction endonuclease is modulated by proteolytic degradation of HsdR subunit of holoenzyme by ClpXP protease, phenomenon known as alleviation of DNA restriction (RA). It was previously shown that UV-induced RA depends on functional recA, recBCD and lexA genes and it has been proposed that chromosomal unmethylated target sequences 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 λ in UV-irradiated cells. We assumed that absence of gene products required for generation of unmethylated DNA would decrease the survival of unmodified λ phage. Our genetic analysis confirmed earlier observation that UV-induced RA depends on excision repair protein UvrA. We show that recF/O/R genes are necessary to induce the SOS response and possibly to stimulate recombination directed replication reaction on DNA substrate prepared by RecBCD enzyme. In addition to helicase activity of RecBCD enzyme, the loading activity of RecA protein by RecBCD is also required for UV-induced RA. PriA, a primosome assembly protein is needed to initiate DNA replication restart and we propose that D-loop is the major recombination intermediate in this process.

EcoKI; lambda; restriction alleviation; recombination; E.coli

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