Abstract
The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein ∼2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-∈M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-∈M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 10400-10404 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 267 |
| Issue number | 15 |
| State | Published - May 25 1992 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
Cite this
The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition. / Menetski, Joseph P.; Varghese, Abraham; Kowalczykowski, Stephen C.
In: Journal of Biological Chemistry, Vol. 267, No. 15, 25.05.1992, p. 10400-10404.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition
AU - Menetski, Joseph P.
AU - Varghese, Abraham
AU - Kowalczykowski, Stephen C.
PY - 1992/5/25
Y1 - 1992/5/25
N2 - The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein ∼2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-∈M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-∈M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.
AB - The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein ∼2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-∈M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-∈M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.
UR - http://www.scopus.com/inward/record.url?scp=0026674169&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026674169&partnerID=8YFLogxK
M3 - Article
C2 - 1350278
AN - SCOPUS:0026674169
VL - 267
SP - 10400
EP - 10404
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -