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PMID: 8026461 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

The EMBO journal ·Vol. 13 ·No. 12 ·1994-06-15 ·Pages 2764-76

Kuzminov A, Schabtach E, Stahl FW

Abstract

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.

MeSH Terms
Base Sequence Cosmids/metabolism DNA Damage DNA, Bacterial/metabolism,ultrastructure DNA-Binding Proteins/metabolism Endodeoxyribonucleases/metabolism Escherichia coli/metabolism Exodeoxyribonuclease V Exodeoxyribonucleases/metabolism Models, Genetic Molecular Sequence Data Nucleic Acid Conformation Protein Binding Rec A Recombinases/metabolism Recombination, Genetic
Chemicals
DNA, Bacterial DNA-Binding Proteins Rec A Recombinases Endodeoxyribonucleases Exodeoxyribonucleases terminase Exodeoxyribonuclease V
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kuzminov A
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Schabtach E
Stahl F W
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1994-06-15
Pages
2764-76
Language
English
Region
England
NLM ID
8208664
PMCID
PMC395156
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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