Abstract

The aim of this research program is to understand in molecular detail the mechanism by which the replication of the herpes simplex virus type 1 genome is initiated and sustained. Our current model is that following circularization of the linear viral genome, theta type replication is initiated at one or more of the three HSV-1 origins, followed by a switch to rolling circle replication. We propose to reconstitute in vitro with purified enzymes whose structure and mechanism we will examine in detail, both the theta and rolling circle phases of HSV-1 DNA replication. We anticipate that these studies will provide us with an insight into the replication of a significant class of human pathogens. The investigation will be organized along the following lines: A. Structure and mechanism of HSV-1 encoded replication enzymes. 1. Origin binding protein (UL9 protein) a. Unwinding of oris by UL9 protein b. Determination of stoichiometry of binding of UL9 protein to Oris c. Interaction of UL9 protein with cellular DNA polymerase alpha-primase d. Determination of three- dimensional structure of UL9 protein 2. DNA polymerase-UL42 protein complex a. Pre-steady state kinetic analysis of enhancement of processivity by UL42 protein b. Effect of phosphorylation of UL42 protein on processivity enhancement 3. Helicase-primase (Primosome) a. Role of UL8 subunit of primosome in its helicase activity b. Analysis of helicase activity c. Analysis of primase activity B. Reconstitution of Oris-dependent DNA replication 1. General approach 2. Examination of specific conditions 3. Transcriptional Activation of transcription C. Further studies of rolling circle DNA replication by a complex of HSV-1 encoded replication enzymes 1. Assembly of complex 2. Mechanism of switch from theta to rolling circle DNA replication.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026538-13
Application #
6170184
Study Section
Biochemistry Study Section (BIO)
Program Officer
Beisel, Christopher E
Project Start
1988-07-01
Project End
2003-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
13
Fiscal Year
2000
Total Cost
$344,112
Indirect Cost

  Institution

Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305

  Publications

Huang, Ke-Jung; Ku, Chia-Chi; Lehman, I Robert (2006) Endonuclease G: a role for the enzyme in recombination and cellular proliferation. Proc Natl Acad Sci U S A 103:8995-9000
Eom, Chi-Yong; Heo, Won Do; Craske, Madeleine L et al. (2004) The neural F-box protein NFB42 mediates the nuclear export of the herpes simplex virus type 1 replication initiator protein (UL9 protein) after viral infection. Proc Natl Acad Sci U S A 101:4036-40
Eom, Chi-Yong; Lehman, I Robert (2003) Replication-initiator protein (UL9) of the herpes simplex virus 1 binds NFB42 and is degraded via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 100:9803-7
Eom, Chi-Yong; Lehman, I Robert (2002) The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to oris, an origin of viral DNA replication. Proc Natl Acad Sci U S A 99:1894-8
He, X; Lehman, I R (2001) An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein. Proc Natl Acad Sci U S A 98:3024-8
He, X; Lehman, I R (2000) Unwinding of a herpes simplex virus type 1 origin of replication (Ori(S)) by a complex of the viral origin binding protein and the single-stranded DNA binding protein. J Virol 74:5726-8
Lee, S S; Lehman, I R (1999) The interaction of herpes simplex type 1 virus origin-binding protein (UL9 protein) with Box I, the high affinity element of the viral origin of DNA replication. J Biol Chem 274:18613-7
Lehman, I R; Boehmer, P E (1999) Replication of herpes simplex virus DNA. J Biol Chem 274:28059-62
Falkenberg, M; Elias, P; Lehman, I R (1998) The herpes simplex virus type 1 helicase-primase. Analysis of helicase activity. J Biol Chem 273:32154-7
Boehmer, P E; Lehman, I R (1997) Herpes simplex virus DNA replication. Annu Rev Biochem 66:347-84

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