IN-VITRO PROCESSING OF HERPES-SIMPLEX VIRUS TYPE-1 DNA-REPLICATION INTERMEDIATES BY THE VIRAL ALKALINE NUCLEASE, UL12

Herpes simplex virus type 1 (HSV-I) DNA replication intermediates exis t in a complex nonlinear structure that does not migrate into a pulsed -held gel, Genetic evidence suggests that the product of the UL12 gene , termed alkaline nuclease, plays a role in processing replication int ermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, J. Virol. 70:2075-2085, 1996). In this study we have tested the hypot hesis that alkaline nuclease acts as a structure-specific resolvase. C ruciform structures generated with oligonucleotides were treated with purified alkaline nuclease; however, instead of being resolved into li near duplexes as would be expected of a resolvase activity, the artifi cial cruciforms were degraded. DNA replication intermediates were isol ated from the well of a pulsed-held gel (''well DNA'') and treated wit h purified HSV-1 alkaline nuclease. Although alkaline nuclease can deg rade virion DNA to completion, digestion of well DNA results in a smal ler-than-unit-length product that migrates as a heterogeneous smear; t his product is resistant to further digestion by alkaline nuclease, Th e smaller-than-unit-length products are representative of the entire H SV genome, indicating that alkaline nuclease is not inhibited at speci fic sequences, To further probe the structure of replicating DNA, well DNA was treated with various known nucleases; our results indicate th at replicating DNA apparently contains no accessible double-stranded e nds but does contain nicks and gaps. Our data suggest that UL12 functi ons at nicks and gaps in replicating DNA to correctly repair or proces s the replicating genome into a form suitable for encapsidation.