Title

The role of the herpes simplex virus type 1 alkaline nuclease in processing DNA replication intermediates

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Microbiology

Degree

Ph.D.

Abstract

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and analysis of this mutant suggests that UL12 plays a role in processing complex DNA replication intermediates. This processing step may be required for the generation of capsids that are competent for egress from the nucleus to the cytoplasm. In order to address the question of whether the AN-1 growth phenotype is due to the loss of UL12 catalytic activity, we constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild type and mutant enzymes from a baculovirus expression system. Neither mutant gene can complement the growth of a UL12 null mutant virus, and neither mutant protein exhibits exonuclease activity. Loss of exonuclease activity therefore correlates with loss of in vivo function. We then tested the hypothesis that alkaline nuclease acts as a structure-specific resolvase. Cruciform structures generated with oligonucleotides were treated with purified alkaline nuclease; however, instead of resolution into linear duplexes as would be expected of a resolvase activity, the artificial cruciforms were degraded. As UL12 is thought to play a role in DNA processing, replication intermediates were isolated from the well of a pulsed-field gel (“well DNA”) and treated with purified HSV-1 alkaline nuclease. Although alkaline nuclease can degrade virion DNA to completion, digestion of well DNA results in a smaller than unit length product that migrates as a heterogeneous smear. To further probe the structure of replicating DNA, well DNA was treated with various known nucleases; our results indicate that replicating DNA apparently contains no accessible double-stranded ends but does contain nicks and gaps. Additionally, electron microscopy reveals an unusual structure at the ends of well DNA. Our data indicate that UL12 functions at nicks and gaps in replicating DNA to correctly repair or process the replicating genome into a form suitable for encapsidation. ^

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