A single mutation, RecB(D1080A), eliminates RecA protein loading but not Chi recognition by RecBCD enzyme

Daniel G. Anderson, Jason J. Churchill, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (χ: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 → Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether χ sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to χ sites by inactivating in a χ-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

Original languageEnglish (US)
Pages (from-to)27139-27144
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number38
DOIs
StatePublished - Sep 17 1999

Fingerprint

Exodeoxyribonuclease V
Rec A Recombinases
Mutation
DNA
Enzymes
Multifunctional Enzymes
Recombinational DNA Repair
Single-Stranded DNA
Genetic Recombination
Escherichia coli
Repair
Switches
Degradation
Processing

ASJC Scopus subject areas

  • Biochemistry

Cite this

A single mutation, RecB(D1080A), eliminates RecA protein loading but not Chi recognition by RecBCD enzyme. / Anderson, Daniel G.; Churchill, Jason J.; Kowalczykowski, Stephen C.

In: Journal of Biological Chemistry, Vol. 274, No. 38, 17.09.1999, p. 27139-27144.

Research output: Contribution to journalArticle

Anderson, Daniel G. ; Churchill, Jason J. ; Kowalczykowski, Stephen C. / A single mutation, RecB(D1080A), eliminates RecA protein loading but not Chi recognition by RecBCD enzyme. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 38. pp. 27139-27144.
@article{d8ea5be0943249ce9e5cb8da5acd408f,
title = "A single mutation, RecB(D1080A), eliminates RecA protein loading but not Chi recognition by RecBCD enzyme",
abstract = "Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (χ: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 → Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether χ sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to χ sites by inactivating in a χ-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.",
author = "Anderson, {Daniel G.} and Churchill, {Jason J.} and Kowalczykowski, {Stephen C.}",
year = "1999",
month = "9",
day = "17",
doi = "10.1074/jbc.274.38.27139",
language = "English (US)",
volume = "274",
pages = "27139--27144",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "38",

}

TY - JOUR

T1 - A single mutation, RecB(D1080A), eliminates RecA protein loading but not Chi recognition by RecBCD enzyme

AU - Anderson, Daniel G.

AU - Churchill, Jason J.

AU - Kowalczykowski, Stephen C.

PY - 1999/9/17

Y1 - 1999/9/17

N2 - Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (χ: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 → Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether χ sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to χ sites by inactivating in a χ-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

AB - Homologous recombination and double-stranded DNA break repair in Escherichia coli are initiated by the multifunctional RecBCD enzyme. After binding to a double-stranded DNA end, the RecBCD enzyme unwinds and degrades the DNA processively. This processing is regulated by the recombination hot spot, Chi (χ: 5'-GCTGGTGG-3'), which induces a switch in the polarity of DNA degradation and activates RecBCD enzyme to coordinate the loading of the DNA strand exchange protein, RecA, onto the single-stranded DNA products of unwinding. Recently, a single mutation in RecB, Asp-1080 → Ala, was shown to create an enzyme (RecB(D1080A)CD) that is a processive helicase but not a nuclease. Here we show that the RecB(D1080A)CD enzyme is also unable to coordinate the loading of the RecA protein, regardless of whether χ sites are present in the DNA. However, the RecB(D1080A)CD enzyme does respond to χ sites by inactivating in a χ-dependent manner. These data define a locus of the RecBCD enzyme that is essential not only for nuclease function but also for the coordination of RecA protein loading.

UR - http://www.scopus.com/inward/record.url?scp=0033578824&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033578824&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.38.27139

DO - 10.1074/jbc.274.38.27139

M3 - Article

C2 - 10480929

AN - SCOPUS:0033578824

VL - 274

SP - 27139

EP - 27144

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 38

ER -