Conformational Dynamics of Bacteriophage T7 DNA Polymerase and its Processivity Factor, Escherichia coli thioredoxin
Journal Article
·
· Proceedings of the National Academy of Sciences of the United States of America
Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.
- Research Organization:
- Brookhaven National Laboratory (BNL) National Synchrotron Light Source
- Sponsoring Organization:
- DOE - OFFICE OF SCIENCE
- DOE Contract Number:
- AC02-98CH10886
- OSTI ID:
- 1019608
- Report Number(s):
- BNL--95453-2011-JA
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 34 Vol. 107; ISSN 0027-8424; ISSN PNASA6
- Country of Publication:
- United States
- Language:
- English
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