Identification and characterization of DNApol and P143 of Autographa californica nuclear polyhedrosis virus /
Prior to this study, the functions of most of the essential DNA replication proteins were inferred from sequence homology alone. With no biochemical proof or characterization of these enzymes, it was impossible to determine what additional proteins might be required, or to formulate a model for the...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2000.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=728331811&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Prior to this study, the functions of most of the essential DNA replication proteins were inferred from sequence homology alone. With no biochemical proof or characterization of these enzymes, it was impossible to determine what additional proteins might be required, or to formulate a model for the mechanism of DNA replication of AcNPV. As a result, we purified and characterized the protein encoded by dnapol, and found it to be a processive enzyme capable of modest strand displacement. The processivity of the core DNA polymerase (DNApol) makes it unlikely that the transient replication assay used to identify essential replication proteins would be sensitive enough to detect a processivity factor. Therefore, it is unlikely that a processivity factor is present among the six proteins identified as essential for DNA replication. DNApol was able to accomplish rolling circle replication in vivo with the assistance of the AcNPV SSB and DNA helicase, suggesting that this may be one of the ways in which AcNPV replicates its genome. Mutagenesis studies had failed to establish P143 as the viral DNA helicase. In order to identify the function of P143, we purified this enzyme from insect cells infected with recombinant virus designed to overexpress P143. ATPase activity co-purified with P143, throughout purification and a gel filtration column. Purified P143 was able to unwind a 40 nucleotide primer annealed to single-stranded M13 DNA in an ATP dependent manner, and support unwinding of a 4 kilobase plasmid in the presence of DNApol and LEF-3, the viral SSB. Glutaraldehyde crosslinking indicated that P143 forms a hexamer in solution. The addition of ATP and MgCl₂ to electrophoretic shift assay reactions decreased P143 DNA binding. Only the nucleotides and divalent cations that support P143 helicase activity affected P143 DNA binding, suggesting that the alteration in DNA binding observed was related to P143 helicase function. Competition assays showed the addition of ATP and MgCl2 increased the rate at which P143 dissociated from DNA. These DNA binding studies suggest that the viral helicase unwinds DNA by a mechanism similar to the inchworm model. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xiii, 153 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 140-152). |