The addAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation

Frederic Chedin, Naofumi Handa, Mark S. Dillingham, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (χ). Recognition of χ triggers attenuation of the 3′- to 5′-nuclease, which permits the generation of recombinogenic 3′-overhanging, single-stranded DNA (ssDNA), terminating at χ. Although the RecBCD enzyme briefly pauses at χ, no specific binding of RecBCD to χ during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate χ sequence (χBs: 5′-AGCGG-3′) during translocation. The binding of AddAB enzyme to the 3′-end of the χBs-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, χBs-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to χBs with high affinity only during translocation. Finally, protection of χBs-specific ssDNA is still observed when translocation occurs in the presence of competitor χBs-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to χBs is an integral part of the AddAB-χBs interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.

Original languageEnglish (US)
Pages (from-to)18610-18617
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number27
DOIs
StatePublished - Jul 7 2006

Fingerprint

Exodeoxyribonuclease V
Single-Stranded DNA
Degradation
Bacilli
Phenol
Bacillus subtilis
Escherichia coli
Adenosine Triphosphate
AddAB enzyme
Enzymes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

The addAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation. / Chedin, Frederic; Handa, Naofumi; Dillingham, Mark S.; Kowalczykowski, Stephen C.

In: Journal of Biological Chemistry, Vol. 281, No. 27, 07.07.2006, p. 18610-18617.

Research output: Contribution to journalArticle

Chedin, Frederic ; Handa, Naofumi ; Dillingham, Mark S. ; Kowalczykowski, Stephen C. / The addAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 27. pp. 18610-18617.
@article{c8f67981418a493584517f8e4da43d25,
title = "The addAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation",
abstract = "The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (χ). Recognition of χ triggers attenuation of the 3′- to 5′-nuclease, which permits the generation of recombinogenic 3′-overhanging, single-stranded DNA (ssDNA), terminating at χ. Although the RecBCD enzyme briefly pauses at χ, no specific binding of RecBCD to χ during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate χ sequence (χBs: 5′-AGCGG-3′) during translocation. The binding of AddAB enzyme to the 3′-end of the χBs-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, χBs-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to χBs with high affinity only during translocation. Finally, protection of χBs-specific ssDNA is still observed when translocation occurs in the presence of competitor χBs-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to χBs is an integral part of the AddAB-χBs interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.",
author = "Frederic Chedin and Naofumi Handa and Dillingham, {Mark S.} and Kowalczykowski, {Stephen C.}",
year = "2006",
month = "7",
day = "7",
doi = "10.1074/jbc.M600882200",
language = "English (US)",
volume = "281",
pages = "18610--18617",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "27",

}

TY - JOUR

T1 - The addAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation

AU - Chedin, Frederic

AU - Handa, Naofumi

AU - Dillingham, Mark S.

AU - Kowalczykowski, Stephen C.

PY - 2006/7/7

Y1 - 2006/7/7

N2 - The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (χ). Recognition of χ triggers attenuation of the 3′- to 5′-nuclease, which permits the generation of recombinogenic 3′-overhanging, single-stranded DNA (ssDNA), terminating at χ. Although the RecBCD enzyme briefly pauses at χ, no specific binding of RecBCD to χ during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate χ sequence (χBs: 5′-AGCGG-3′) during translocation. The binding of AddAB enzyme to the 3′-end of the χBs-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, χBs-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to χBs with high affinity only during translocation. Finally, protection of χBs-specific ssDNA is still observed when translocation occurs in the presence of competitor χBs-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to χBs is an integral part of the AddAB-χBs interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.

AB - The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (χ). Recognition of χ triggers attenuation of the 3′- to 5′-nuclease, which permits the generation of recombinogenic 3′-overhanging, single-stranded DNA (ssDNA), terminating at χ. Although the RecBCD enzyme briefly pauses at χ, no specific binding of RecBCD to χ during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate χ sequence (χBs: 5′-AGCGG-3′) during translocation. The binding of AddAB enzyme to the 3′-end of the χBs-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, χBs-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to χBs with high affinity only during translocation. Finally, protection of χBs-specific ssDNA is still observed when translocation occurs in the presence of competitor χBs-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to χBs is an integral part of the AddAB-χBs interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.

UR - http://www.scopus.com/inward/record.url?scp=33745838577&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33745838577&partnerID=8YFLogxK

U2 - 10.1074/jbc.M600882200

DO - 10.1074/jbc.M600882200

M3 - Article

C2 - 16632468

AN - SCOPUS:33745838577

VL - 281

SP - 18610

EP - 18617

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 27

ER -