Temperature-sensitive mutants of the EcoRI endonuclease

Muir, R S; Flores, Humberto; Zinder, Norton D.; Model, Peter; Soberón, Xavier; Heitman, Joseph

doi:10.1006/jmbi.1997.1419

Cited by 17 publications

(11 citation statements)

References 63 publications

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“…These strains also carry an SOS:: lacZ fusion that enabled us to monitor SOS induction by scoring for blue colony formation on Xgal indicator medium. As with other Eco RI TS mutants (Heitman et al ., 1989; Muir et al ., 1997), the R200K and E144C mutants induced the SOS response (see Table 1) and this induction required both RecA and RecB and was absent in the lexA3 mutant strain (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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

Heitman

Ivanenko

Kiss

1999

Molecular Microbiology

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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.

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Section: Resultsmentioning

confidence: 99%

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

Heitman

Ivanenko

Kiss

1999

Molecular Microbiology

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“…23 Although the R56Q mutant enzyme showed activity similar to that of the wild-type enzyme at permissive temperature, 46 we did not want a secondary mutation to confound subsequent biochemical experiments on the promiscuous mutant proteins. We therefore amplified the wild-type EcoRI endonuclease gene from the plasmid pMB3, 47 and inserted it into pET24a (Novagen) between the NheI and BamHI sites to create pPS12, in which transcription of the endonuclease gene is directed by the bacteriophage T7 promoter.…”

Section: Site-directed Mutagenesismentioning

confidence: 95%

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

Sapienza

Torre

McCoy

et al. 2005

Journal of Molecular Biology

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“…The temperature sensitivity of SMEG35 could result from thermolability of the mutant protein, a general folding defect (as described previously for EcoRI endonuclease and phage P22 tail spike endorhamnosidase [9,15]), or a general reduction in specific activity resulting in a mutant protein that cannot keep up with the increased metabolic demands of growth at higher temperatures. To address this, the wild-type and mutant DdlA proteins were purified and tested for enzymatic activity at different temperatures.…”

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Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

2000

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A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and Dcycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.A nearly universal component of bacterial cell walls is peptidoglycan, a macromolecule that is composed of polysaccharide chains that are cross-linked by short peptide bridges. Peptidoglycan gives the bacterial cell its characteristic shape and prevents the cell from lysing due to high internal osmotic pressure. Our understanding of how peptidoglycan is synthesized in bacteria is derived mostly from work done with Escherichia coli in which a number of temperature-sensitive mutants have been isolated that are defective in the biosynthesis of peptidoglycan at 42°C (10,11,13,14,20). Two hallmarks of these mutants are cell lysis at the nonpermissive temperature and suppression of the temperature-sensitive phenotype on media containing osmotic stabilizers (10,11,13,14,20).We previously described the generation of a bank of temperature-sensitive mutants of Mycobacterium smegmatis mc 2 155 (2, 4). One of the mutants, SMEG35, exhibits the two phenotypic characteristics associated with E. coli mutants defective in peptidoglycan biosynthesis. First, SMEG35 cells grown at 30°C to an optical density at 600 nm of 0.5 and then shifted to 42°C lyse after one doubling time, as evidenced by a visual clearing of the culture and the appearance of flocculent material (data not shown). Second, the temperature-sensitive phenotype of SMEG35 can be suppressed on growth medium containing either 0.5 M sucrose or 0.2 M NaCl (data not shown).The bacterial strains and plasmids used in this study are listed in Table 1. To identify the mutated gene, SMEG35 was complemented with an M. smegmatis genomic cosmid library as previously described (2). A sublibrary was constructed from the complementing cosmid pAEB222 and the E. coli-mycobacterial shuttle vector pMD30 as described previously (2). The nucleotide sequence was determined for the insert of the smallest complementing subclone pAEB224 with an ABI310 automated sequencer (PE Biosystems, Foster City, Calif.) and genespecific primers. The presence of a mutation in the ddlA gene of SMEG35 was confirmed by sequencing of the mutant allele. The gene was amplified from the SMEG35 genome with Pfu DNA polymerase (Stratagene) and the primers 5Ј-TTGTGACTGCCCC GAACC-3Ј (forward) and 5Ј-CGAAAAACCCGTCGAGCC-3Ј (reverse) in a PCR mixture supplemented with 5% formamide. Sequence analysis revealed a single mutation at bp 1095 of ddlA that changes a C to a T on the top strand. This mutation results in an alanine-to-valine substitution at amino acid 365 of Ddl, close to the C terminus of the protein. The alanine that is mutated in the SMEG35 Ddl is a nonconserved amino acid residue that does not correspond to any of the amino acids that were previously shown to be...

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Temperature-sensitive mutants of the EcoRI endonuclease

Cited by 17 publications

References 63 publications

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

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

Thermodynamic and Kinetic Basis for the Relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI Endonucleases

Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a ddlA Mutant of Mycobacterium smegmatis

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