期刊
JOURNAL OF VIROLOGY
卷 85, 期 2, 页码 968-978出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.01690-10
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资金
- NIH [AI-21747, NS15190, AI-059764, GM-073832, 1S10RR022624-01]
- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR022624] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI059764, R37AI021747, R01AI021747] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM073832] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS015190, R56NS015190] Funding Source: NIH RePORTER
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.
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