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Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.

ABSTRACT: BACKGROUND: recD, located between recB and argA, encodes the smallest polypeptide (60 kDa) of the heterotrimeric enzyme RecBCD in Escherichia coli. RecD is a 5'-3' helicase and is required for the nuclease activity of RecBCD and for tight binding to dsDNA ends. Here, we have tested the hypothesis that RecD regulates the structure and activities of RecBCD, including RecA loading. RESULTS: To characterize its regulatory functions, recD was genetically fused to recB through deletion and substitution mutations. The recB-recD fusion led to a decreased amount of the heterotrimer. Both fusion mutants proved to be recombination proficient, viable and resistant to DNA damaging agents, and to have DNA unwinding, ATP-dependent dsDNA exonuclease and Chi genetic activities. CONCLUSION: Our findings suggest that the recB-recD fusion may form a RecBD fusion protein and therefore affect RecD assembly, but this does not change the three-dimensional structure of the heterotrimer.

SUBMITTER: Portakal O

PROVIDER: S-EPMC2586629 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.

Portakal Oytun O Doğan Pakize P

BMC biochemistry 20081010

<h4>Background</h4>recD, located between recB and argA, encodes the smallest polypeptide (60 kDa) of the heterotrimeric enzyme RecBCD in Escherichia coli. RecD is a 5'-3' helicase and is required for the nuclease activity of RecBCD and for tight binding to dsDNA ends. Here, we have tested the hypothesis that RecD regulates the structure and activities of RecBCD, including RecA loading.<h4>Results</h4>To characterize its regulatory functions, recD was genetically fused to recB through deletion an ...[more]

PMID: 18847457

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Project description:A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

Dataset Information

Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.

Publications

Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli.

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