Analysis of the DNA-binding domain of the HSV-1 origin-binding protein.
In order to understand DNA-protein interactions at the origin of DNA replication in herpes simplex virus type 1 (HSV-1), we have undertaken an analysis of the DNA-binding domain of the origin-binding protein (OBP) and its mechanism of binding to the Oris sequence of HSV-1. Mutant DNA-binding domains were constructed, expressed in vitro, and used to test for binding by gel shift analysis. A C-terminal deletion mutant was functional in binding, thereby redefining the C-terminal boundary of the DNA-binding domain at amino acid 822. Fifteen insertion mutants were also constructed across the DNA-binding domain. Several of these mutants were unable to bind DNA. Interestingly, 4 mutants that destroy DNA binding fall within a region that has a particularly high degree of sequence similarity to the varicella zoster virus gene 51 product. A second objective was to define how the DNA-binding domain interacts with the origin. Results of gel shift analysis using contranslated proteins of different sizes suggest that the DNA-binding domain can interact with a single binding site as a monomer. Binding to the wild-type Oris template indicated that the binding domains can interact with both binding sites I and II independent of any cooperative effect mediated by the amino-termini. This suggests that the basic unit of recognition involved in OBP/Oris interactions may contain a single DNA-binding domain of OBP in association with a single binding site.
Martin
D W
DW
Department of Microbiology, University of Texas Health Science Center at San Antonio 78284-7758.
Muñoz
R M
RM
Oliver
D
D
Subler
M A
MA
Deb
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012045:Regulatory Sequences, Nucleic Acid; D014764:Viral Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C055789:origin-binding proteins, viral