Conformational changes induced in herpes simplex virus DNA polymerase upon DNA binding.
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.
Weisshart
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K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Kuo
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Painter
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Wright
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Furman
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Coen
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D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D005470:Fluorometry; D015335:Molecular Probes; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D015335:Molecular Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D004257:DNA Polymerase II; D014357:Trypsin