期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1993
卷号：90
期号：3
页码：1028-1032
DOI：10.1073/pnas.90.3.1028
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Herpesvirus DNA polymerases are prototypes for alpha-like DNA polymerases and important targets for antiherpesvirus drugs. We have investigated changes in the catalytic subunit of herpes simplex virus DNA polymerase following DNA binding by using the techniques of endogeneous fluorescence quenching and limited proteolysis. The fluorescence studies revealed a reduction in the rate of quenching by acrylamide in the presence of DNA without changes in the wavelength of the emission peak or in the lifetime of the fluorophore, consistent with the possibility of conformational changes. Strikingly, the proteolysis studies revealed that binding to a variety of natural and synthetic DNA and RNA molecules induced the appearance of a new cleavage site for trypsin near residue 1060 of the protein and increased cleavage by trypsin near the center of the protein. The extent of these cleavages correlated with the affinity of the polymerase for these ligands. These data provide strong evidence that binding to nucleic acid polymers induces substantial localized conformational changes in the polymerase. The locations of enhanced tryptic cleavage near sites implicated in substrate recognition and interaction with a processivity factor suggest that the conformational changes are important for catalysis and processivity of this prototype alpha-like DNA polymerase. Inhibition of these changes may provide a mechanism for antiherpesvirus drugs.
