Herpes simplex virus type 1 helicase-primase: DNA binding and consequent protein oligomerization and primase activation.
The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.
Department of Molecular Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06085, USA.
Yan
Y
Chen
Ping
P
Bai
Shannon
S
Mackay
George
G
Korza
John H
JH
Carson
Robert D
RD
Kuchta
Sandra K
SK
Weller
D000818:Animals; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D011485:Protein Binding; D055503:Protein Multimerization; D020349:Surface Plasmon Resonance; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D014764:Viral Proteins; D004247:DNA; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases