Biochemical basis of the constitutive coprotease activity of RecA P67W protein

Julie K. Mirshad, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.

Original languageEnglish (US)
Pages (from-to)5937-5944
Number of pages8
JournalBiochemistry
Volume42
Issue number19
DOIs
StatePublished - May 20 2003

Fingerprint

Rec A Recombinases
Single-Stranded DNA
DNA
Mutant Proteins
Proteins
Genetic Recombination
Nucleic Acid Heteroduplexes
Escherichia coli Proteins
Protein Binding
Escherichia coli
Joints
Binding Sites
Phenotype
Defects
Mutation
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biochemical basis of the constitutive coprotease activity of RecA P67W protein. / Mirshad, Julie K.; Kowalczykowski, Stephen C.

In: Biochemistry, Vol. 42, No. 19, 20.05.2003, p. 5937-5944.

Research output: Contribution to journalArticle

Mirshad, Julie K. ; Kowalczykowski, Stephen C. / Biochemical basis of the constitutive coprotease activity of RecA P67W protein. In: Biochemistry. 2003 ; Vol. 42, No. 19. pp. 5937-5944.
@article{d89a1bf4711646848e62c091d2db0517,
title = "Biochemical basis of the constitutive coprotease activity of RecA P67W protein",
abstract = "The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.",
author = "Mirshad, {Julie K.} and Kowalczykowski, {Stephen C.}",
year = "2003",
month = "5",
day = "20",
doi = "10.1021/bi027232q",
language = "English (US)",
volume = "42",
pages = "5937--5944",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "19",

}

TY - JOUR

T1 - Biochemical basis of the constitutive coprotease activity of RecA P67W protein

AU - Mirshad, Julie K.

AU - Kowalczykowski, Stephen C.

PY - 2003/5/20

Y1 - 2003/5/20

N2 - The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.

AB - The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.

UR - http://www.scopus.com/inward/record.url?scp=0038628984&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0038628984&partnerID=8YFLogxK

U2 - 10.1021/bi027232q

DO - 10.1021/bi027232q

M3 - Article

C2 - 12741852

AN - SCOPUS:0038628984

VL - 42

SP - 5937

EP - 5944

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 19

ER -