DNA nicks inflicted by restriction endonucleases are repaired by a RecA‐ and RecB‐dependent pathway in Escherichia coli

Abstract

SummaryTwo mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of in¯icting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at noncognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.

Single-strand interruptions in replicating chromosomes cause double-strand breaks

“…Rather, the 5Ј-end of the nick is likely to be bound by the M13 nicking enzyme [may be covalently (48)], which creates a certain ''microenvironment'' around the nick and likely even around the subsequent double-strand end. Therefore, nicks introduced by other means [for example, by ''nicking'' mutants of restriction endonucleases (49,50)] could better approximate spontaneous single-strand interruptions encountered by replication forks in vivo.…”

The isolation of strand-specific nicking endonucleases from a randomized SapI expression library

“…The first step of the method is transformation of the library into bacterial cells where the genomic DNA is not protected by DNA methylation of SapI sites. In this step, a stringent selection occurs since RecA + , Lig + strains are significantly more tolerant of single-strand nicks as compared to double-strand cuts (13). In this genetic selection, survivors are expected to be expressing nicking variants, low activity variants and null variants.…”

“…The four SapI variants with top-strand nicking activity contain amino acid substitutions within or near a sequence which conforms to the consensus PD(X) [8][9][10][11][12][13][14][15][16][17][18][19][20] D/EXK for Type II catalytic centers. Thus, the amino acids E250, E263 and K265 may form an active site for bottom-strand cleavage.…”

“…The SapI recognition site is in bold. variants [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. All five positive clones (2,9,11,15,18) were sequenced to determine the responsible amino acid substitutions.…”

“…Random mutagenesis of EcoRI also yielded some variants that preferentially nick DNA. For example, the EcoRI R200C and R200K variants generate a higher proportion of nicked circular DNA than does the wt EcoRI enzyme (12,13).…”