DNA-Thumb Interactions and Processivity of T7 DNA Polymerase in Comparison to Yeast Polymerase Î
The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized. The crystal structure of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme i...
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Published in | The Journal of biological chemistry Vol. 279; no. 18; p. 18288 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
American Society for Biochemistry and Molecular Biology
30.04.2004
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Online Access | Get full text |
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Summary: | The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized. The crystal structure
of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme interacts with the 3â²-end of the primer-template,
but does not show how thioredoxin confers processivity to the polymerase. In the crystal structure highly conserved amino
acids Asn 335 and Ser 338 of the thumb subdomain of T7 DNA polymerase are seen to interact with phosphates 7 and 8 of the DNA template strand. Results
with a mutant T7 DNA polymerase in which aliphatic residues are substituted for these amino acids and experiments with different
length and methylphosphonate-modified primer-templates demonstrate that these interactions are essential for processive synthesis
and d(A·T) n tract bypass. Our data with methylphosphonate-modified DNA suggests that thioredoxin confers processivity to T7 DNA polymerase
in part by causing an interaction with the phosphate backbone or minor groove of DNA. Residues Asn 335 and Ser 338 may also function with a nearby helix-loop-helix motif located at residues 339â372 to enclose the DNA during processive synthesis.
Our results suggest that this structure must be held close to the DNA by ionic interactions to function. These interactions
also allow for DNA sliding but physically block the passage of a 3T bulge in the template. In contrast, yeast polymerase η,
a polymerase that non-mutagenically repairs cis - syn thymidine dimers, allows the same bulge to slide past its thumb subdomain during synthesis. A relaxed thumb interaction with
the DNA could account for the notably low processivity of polymerase η. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M400282200 |