Identification of sbcD mutations as cosuppressors of recBC that allow propagation of DNA palindromes in Escherichia coli K-12

Abstract

The function of an open reading frame (orf-45) located upstream of the sbcC gene of Escherichia coli was investigated. Mutations that inactivate sbcC improve the ability to propagate Ared gam phage that carry a palindromic sequence in their DNA. They also act with sbcB mutations as cosuppressors of the defects in recombination, DNA repair, and cell viability associated with recBC mutations. A 1,282-bp cassette encoding resistance to kanamycin was used to disrupt orf-45. The mutation, which has a polar effect on the expression of sbcC, allowed stable propagation of palindromic k phage even when the sbcC gene product was provided in trans. Additional nonpolar mutations in orf-45 were isolated on the basis of their ability to improve the growth of recBC sbcB strains. These mutations also confer resistance to mitomycin C, allow efficient recombination in Hfr crosses, and facilitate stable propagation of palindromic phage. It is concluded that the products of orf-45 and sbcC are functionally related. The orf-45 gene is therefore renamed sbcD.The sbcC gene of Escherichia coli was defined originally by mutations that arise spontaneously in recBC sbcB strains (16). These mutations act with mutations in sbcB to suppress the defects in recombination and DNA repair associated with the recBC genotype and are selected, therefore, during normal growth because of the improved viability. Subsequently, sbcC single mutants were shown to be good hosts for a Xred gam phage carrying a DNA sequence with palindromic symmetry (Xpal) (5). Phage of this type grow poorly in sbcC+ cells. Leach and Stahl (13) had observed this feature of the sbcC phenotype in their earlier work with recBC sbcB sbcC strains but, because isogenic strains were not constructed and used for comparisons, had attributed it to the absence of exonuclease V (RecBCD enzyme) and exonuclease I (the product of sbcB). This seemed reasonable since palindromes might form hairpins or cruciforms which could be recognized and cleaved by nucleases that normally resolve Holliday intermediates in genetic recombination. Kulkarni and Stahl (11) MATERIALS AND METHODSBacterial strains and X phages. The E. coli K-12 strains and A phages used are described in Table 1. Phage stocks were prepared on strain JC7623, which allows stable propagation of the 571-bp palindrome carried by XMMS1632.Plasmids. Plasmid constructs carrying DNA inserts from the sbcC-orf-45 region of the chromosome are shown in Fig. 1. pSB43, pJP77, and pIN510 are described in detail elsewhere (3,23,28). pIN507 corresponds to pJP77 sbcC:: TnlOOO no. 5 described by Naom et al. (23). pFG103 was made by cloning a BamHI-HindIII fragment of approximately 14.5 kb from pJP77 into pACYC184 (6). A 1,282-bp cassette (kan) encoding resistance to kanamycin was cloned from pUC4K (29) into the unique PstI site of pFG103 to give pFG103K. pFG106 was constructed by removing the Kmr insertion from pFG103K by digestion with EcoRI and inserting it into the EcoRI site of the temperature-sensitive replicon pHSG415 (10). The sbcC+ construc...

Role ofRAD52Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

“…The mre11-H125N and rad50S mutations conferred the same phenotype as did the sae2 mutation in this assay, again suggesting that the Mre11 nuclease and Sae2 resolve palindromes. The results of these two studies are consistent with the observation that palindromes are stabilized in sbcC and sbcD mutants of E. coli (49,115).…”

Human Rad50 Is Physically Associated with Human Mre11: Identification of a Conserved Multiprotein Complex Implicated in Recombinational DNA Repair

“…SbcC functions in complex with a coordinately regulated protein, SbcD, to mediate ATP-dependent exonuclease activity (12a, 40). It is interesting that the three known Mre11 homologs exhibit significant homology to the SbcD proteins from E. coli and Bacillus subtilis (23,39,43). Similarity between SbcC/SbcD and ScRad50/ScMre11 has been noted previously (72).…”

The role of SOS and flap processing in microsatellite instability in Escherichia coli

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