Abstract
The apparent DNA site size obtained from an assay monitoring the ATPase activity of Escherichia coli recA protein (n = 3.5) differs from that determined from a direct DNA binding assay (n = 7) done under identical conditions. Investigation of this discrepancy indicates that at a DNA:protein ratio of 3.5:1, one-half of the recA protein population is less sensitive to ATPase activity inhibition by the nonhydrolyzable ATP analogue adenosine 5′-O-(3-thiotriphosphate) (ATPγS), suggesting that the recA protein filament is asymmetric with respect to NTP affinity. This asymmetry does not depend on the presence of ATPγS since the apparent Km for ATP derived from single-stranded DNA-dependent ATP hydrolysis activity is dependent on the DNA:protein ratio. Three models are proposed to account for the observed site size discrepancy and the NTP binding affinity asymmetry. They differ mainly in the intrinsic site size for each recA protein monomer and in the number of DNA-binding sites/recA molecule. Gel filtration of recA-single-stranded DNA complexes at different DNA:protein ratios complements the enzymological data and provides an additional method of distinguishing among the proposed models. The phenomenon of subunit nonequivalence within the recA protein presynaptic filament may provide a molecular basis for understanding how recA protein can discriminate between different DNA molecules during homologous pairing.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 5450-5458 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 266 |
| Issue number | 9 |
| State | Published - 1991 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Asymmetry in the recA protein-DNA filament. / Lauder, Scott D.; Kowalczykowski, Stephen C.
In: Journal of Biological Chemistry, Vol. 266, No. 9, 1991, p. 5450-5458.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Asymmetry in the recA protein-DNA filament
AU - Lauder, Scott D.
AU - Kowalczykowski, Stephen C.
PY - 1991
Y1 - 1991
N2 - The apparent DNA site size obtained from an assay monitoring the ATPase activity of Escherichia coli recA protein (n = 3.5) differs from that determined from a direct DNA binding assay (n = 7) done under identical conditions. Investigation of this discrepancy indicates that at a DNA:protein ratio of 3.5:1, one-half of the recA protein population is less sensitive to ATPase activity inhibition by the nonhydrolyzable ATP analogue adenosine 5′-O-(3-thiotriphosphate) (ATPγS), suggesting that the recA protein filament is asymmetric with respect to NTP affinity. This asymmetry does not depend on the presence of ATPγS since the apparent Km for ATP derived from single-stranded DNA-dependent ATP hydrolysis activity is dependent on the DNA:protein ratio. Three models are proposed to account for the observed site size discrepancy and the NTP binding affinity asymmetry. They differ mainly in the intrinsic site size for each recA protein monomer and in the number of DNA-binding sites/recA molecule. Gel filtration of recA-single-stranded DNA complexes at different DNA:protein ratios complements the enzymological data and provides an additional method of distinguishing among the proposed models. The phenomenon of subunit nonequivalence within the recA protein presynaptic filament may provide a molecular basis for understanding how recA protein can discriminate between different DNA molecules during homologous pairing.
AB - The apparent DNA site size obtained from an assay monitoring the ATPase activity of Escherichia coli recA protein (n = 3.5) differs from that determined from a direct DNA binding assay (n = 7) done under identical conditions. Investigation of this discrepancy indicates that at a DNA:protein ratio of 3.5:1, one-half of the recA protein population is less sensitive to ATPase activity inhibition by the nonhydrolyzable ATP analogue adenosine 5′-O-(3-thiotriphosphate) (ATPγS), suggesting that the recA protein filament is asymmetric with respect to NTP affinity. This asymmetry does not depend on the presence of ATPγS since the apparent Km for ATP derived from single-stranded DNA-dependent ATP hydrolysis activity is dependent on the DNA:protein ratio. Three models are proposed to account for the observed site size discrepancy and the NTP binding affinity asymmetry. They differ mainly in the intrinsic site size for each recA protein monomer and in the number of DNA-binding sites/recA molecule. Gel filtration of recA-single-stranded DNA complexes at different DNA:protein ratios complements the enzymological data and provides an additional method of distinguishing among the proposed models. The phenomenon of subunit nonequivalence within the recA protein presynaptic filament may provide a molecular basis for understanding how recA protein can discriminate between different DNA molecules during homologous pairing.
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UR - http://www.scopus.com/inward/citedby.url?scp=0025951863&partnerID=8YFLogxK
M3 - Article
C2 - 1826000
AN - SCOPUS:0025951863
VL - 266
SP - 5450
EP - 5458
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 9
ER -