Abstract
RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced ∼ 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at χ sites, which are hotspots for genetic recombination. Since this interaction with χ significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 605-620 |
| Number of pages | 16 |
| Journal | Journal of Molecular Biology |
| Volume | 231 |
| Issue number | 3 |
| State | Published - 1993 |
| Externally published | Yes |
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Keywords
- ATP-dependent nuclease
- DNA helicase
- Genetic recombination
- recBCD enzyme
ASJC Scopus subject areas
- Virology
Cite this
Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks χ-specific, but not non-specific, nuclease activity. / Eggleston, Angela K.; Kowalczykowski, Stephen C.
In: Journal of Molecular Biology, Vol. 231, No. 3, 1993, p. 605-620.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks χ-specific, but not non-specific, nuclease activity
AU - Eggleston, Angela K.
AU - Kowalczykowski, Stephen C.
PY - 1993
Y1 - 1993
N2 - RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced ∼ 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at χ sites, which are hotspots for genetic recombination. Since this interaction with χ significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.
AB - RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced ∼ 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at χ sites, which are hotspots for genetic recombination. Since this interaction with χ significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.
KW - ATP-dependent nuclease
KW - DNA helicase
KW - Genetic recombination
KW - recBCD enzyme
UR - http://www.scopus.com/inward/record.url?scp=0027240259&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027240259&partnerID=8YFLogxK
M3 - Article
C2 - 8390577
AN - SCOPUS:0027240259
VL - 231
SP - 605
EP - 620
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 3
ER -