Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase.

The herpes helicase-primase (UL5-UL8-UL52) very inefficiently unwinds double-stranded DNA. To better understand the mechanistic consequences of this inefficiency, we investigated protein displacement activity by UL5-UL8-UL52, as well as the impact of coupling DNA synthesis by the herpes polymerase with helicase activity. While the helicase can displace proteins bound to the lagging strand template, bound proteins significantly impede helicase activity. Remarkably, UL5-UL8-UL52, an extremely inefficient helicase, disrupts the exceptionally tight interaction between streptavidin and biotin on the lagging strand template. It also unwinds DNA containing streptavidin bound to the leading strand template, although it does not displace the streptavidin. These data suggest that the helicase may largely or completely wrap around the lagging strand template, with minimal interactions with the leading strand template. We utilized synthetic DNA minicircles to study helicase activity coupled with the herpes polymerase-processivity factor (UL30-UL42). Coupling greatly enhances unwinding of DNA, although bound proteins still inhibit helicase activity. Surprisingly, while UL30-UL42 and two noncognate polymerases (Klenow Fragment and T4 DNA polymerase) all stimulate unwinding of DNA by the helicase, the isolated UL30 polymerase (i.e., no UL42 processivity factor) binds to the replication fork but in a manner that is incompetent in terms of coupled helicase-polymerase activity.