Date of Award
Doctor of Philosophy (PhD)
Chemistry & Biochemistry
Robert D. Kuchta
Jennifer F. Kugel
Deborah S. Wuttke
Carlos E. Catalano
Dylan J. Taatjes
There are seven proteins essential for Herpes Simplex Virus Type 1 (HSV-1) DNA replication. While some of their roles are known, many of their mechanisms remain unclear. Understanding the mechanism by which herpes replicates its viral genome may provide insight into targeting critical pathways to more effectively treat infection.
This work identifies new roles and mechanisms of the essential enzymes at the herpes replication fork: the helicase-primase (UL5-UL8-UL52) and polymerase-processivity factor (UL30-UL42). I determined that as with other helicases, herpes helicase-primase displays varied ability to displace different bound proteins. Though the polymerase-processivity factor enhances helicase activity considerably, bound proteins still greatly impede unwinding during coupled DNA replication. From streptavidin displacement studies, I propose a model where the monomeric helicase-primase may largely or completely encircle the lagging strand template much like multimeric helicases which form rings around the DNA.
I exploited a variety of minicircle DNA substrates to determine how the herpes helicase-primase and polymerase-processivity factor function together during DNA replication. I determined that herpes replication machinery effectively utilizes noncognate enzymes, i.e. the herpes polymerase-processivity factor utilizes E. coli DnaB helicase to synthesize longer products. However, the cognate enzyme pair is more efficient, suggesting tight coordination. Though UL30-UL42 stimulates DNA unwinding by the helicase-primase, UL30-UL42 does not stimulate the ATPase activity of the helicase-primase. Continuing my investigation of coupling at the replication fork, I found that the leading and lagging strand polymerases are not tightly coupled; i.e., the inhibition of lagging strand DNA synthesis does not impact leading strand synthesis.
The processivity factor, UL42, enhances the processivity of the polymerase, UL30, by a factor of 8 in an all-or-nothing manner via a contact 24 nucleotides upstream of the primer 3’-terminus. In the absence of the processivity factor, the polymerase is essentially nonfunctional at the replication fork under conditions where the helicase must unwind the DNA in front of the polymerase. While the polymerase can function with a helicase lacking the UL8 subunit, UL8 increases the length and number of DNA products synthesized during helicase-polymerase coupled DNA replication. I discuss in detail the potential roles that UL42 and UL8 play to achieve efficient herpes replication.
Dickerson, Sarah Michelle, "Interplay Between the Helicase-Primase and the Dna Polymerase-Processivity Factor During Herpes Dna Replication" (2017). Chemistry & Biochemistry Graduate Theses & Dissertations. 239.