Functional characterization of Marek's disease virus (MDV) origin-binding protein (OBP): analysis of its origin-binding properties.
In previous studies, we identified a Marek's disease virus (MDV) origin-binding protein (OBP) gene that is highly homologous to the herpes simplex virus type 1 UL9 gene that encodes an OBP and functions as an initiator protein for viral DNA replication. In this study, a protein of 95 kDa was produced in coupled in vitro transcription-translation reaction with the plasmid containing the wild type MDV OBP gene. The in vitro synthesized protein was detected by immunoprecipitation with a penta-histidine specific monoclonal antibody. Further characterization of MDV OBP was accomplished using electrophoretic mobility shift assay (EMSA) with the in vitro expressed MDV OBP using a double-stranded (ds) 26-mer oligonucleotide as the probe, which was designed from the putative MDV OBP binding site present in the serotype 1 or 2 MDV replication origin. The EMSA results indicated that MDV OBP could form a protein-DNA complex with the ds 26-mer oligonucleotide designed from serotype 1 or 2 replication origin. A series of 26-mer oligonucleotides with two-base-pair (bp) substitution across the putative MDV OBP binding site were used in competitive EMSA to determine the recognition sequence for the MDV OBP. The results demonstrated that the recognition sequence for MDV OBP was the TTCGCACC that is a subset of a 9-bp element (CGTTCGCAC) conserved in the replication origins of alphaherpesviruses. Furthermore, the results of EMSA with a series of deletion mutants from the N-terminus of MDV OBP indicated that the origin-binding domain was located at the amino acids region 528 to 841 of the wild-type MDV OBP. Taken together, our results suggest that the MDV OBP gene encodes an OBP of MDV.
Wu
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TF
Department of Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan. wutingfe@ms65.hinet.net
Chen
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Wu
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D025922:Mardivirus / Q000502:physiology*; D008969:Molecular Sequence Data; D018741:Replication Origin; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C055789:origin-binding proteins, viral