SbcCD Causes a Double-Strand Break at a DNA Palindrome in the Escherichia coli Chromosome

Abstract

Long DNA palindromes are sites of genome instability (deletions, amplification, and translocations) in both prokaryotic and eukaryotic cells. In Escherichia coli, genetic evidence has suggested that they are sites of DNA cleavage by the SbcCD complex that can be repaired by homologous recombination. Here we obtain in vivo physical evidence of an SbcCD-induced DNA double-strand break (DSB) at a palindromic sequence in the E. coli chromosome and show that both ends of the break stimulate recombination. Cleavage is dependent on DNA replication, but the observation of two ends at the break argues that cleavage does not occur at the replication fork. Genetic analysis shows repair of the break requires the RecBCD recombination pathway and PriA, suggesting a mechanism of bacterial DNA DSB repair involving the establishment of replication forks.

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

“…We tested ExoI (encoded by xonA), which attacks 39 ends (Lehman and Nussbaum 1964); RecJ, which attacks 59 ends (Lovett and Kolodner 1989); and ExoVII (encoded by xseA), which can target either end (Chase and Richardson 1974). We also tested the SbcCD enzyme, which has multiple nuclease activities, including the ability to remove 39 overhangs from partial duplexes and to cut hairpin structures (Chalker et al 1988;Connelly et al 1998Connelly et al , 1999Eykelenboom et al 2008). Synthetic lethality between the covered and the uncovered mutations is revealed if the construct fails to show growth of plasmid-free Lac À clones (white colonies and white sectors within blue colonies) on agar plates supplemented with X-gal and IPTG .…”

DNA tandem repeat instability in the Escherichia coli chromosome is stimulated by mismatch repair at an adjacent CAG·CTG trinucleotide repeat

“…1 A and B). This tandem repeat cassette was previously used to show that a DSB caused by cleavage of a 246-bp palindrome in lacZ by the hairpin endonuclease SbcCD can result in recombination between the two zeocin repeats to form an intact zeocin resistance gene (15). As shown in Fig.…”

“…The first hypothesis tested was that a single strand of the TNR array was able to form a hairpin-like structure that was cleaved by SbcCD to generate a DSB, as was previously shown to occur at the site of a DNA palindrome (15). The second hypothesis was that a doublestrand end was generated at the site of the TNR array by replication fork reversal.…”

“…Furthermore, the DSB generated by this cleavage stimulates recombination at a 275-bp direct repeat located 6.3 kb away in the cynX gene (15). This result provided us with a sensitive assay for the detection of DSB formation at the site of expanded CAG·CTG TNR arrays inserted in the place of the palindrome at the lacZ locus.…”

“…It was recently established that a 246-bp DNA palindrome inserted at the lacZ locus in the E. coli chromosome is cleaved by the hairpin endonuclease SbcCD (the homolog of Rad50/Mre11 in eukaryotes) to generate a double-strand break (DSB) (15). Furthermore, the DSB generated by this cleavage stimulates recombination at a 275-bp direct repeat located 6.3 kb away in the cynX gene (15).…”