In vitro expression of the human cytomegalovirus DNA polymerase gene: effects of sequence alterations on enzyme activity.
Genomic DNA of the Towne strain human cytomegalovirus polymerase (pol) gene (4.4-kb RsrII-NcoI segment of the EcoRI J fragment) was cloned into plasmids containing either the T3 or the T7 promoter for in vitro transcription-translation studies. The translation efficiency of unmodified pol cRNA was poor in this system and could not be improved by capping. However, the efficiency could be enhanced by replacing the leader sequence with a 40-bp AT-rich sequence derived from an alfalfa mosaic virus, R4. pol cRNA directed the synthesis of a 140-kDa polypeptide in a rabbit reticulocyte translation system. The in vitro-translated wild-type enzyme possessed significant polymerization activity which could be stimulated by salt as could that of the authentic enzyme purified from virus-infected cells. To study the critical domains of this enzyme, nine mutations were introduced into the pol gene around the conserved domains of eukaryotic polymerase by oligonucleotide-directed mutagenesis. Two constructs with mutations at amino acid residues 323 to 325 (M32QS) and 725 to 726 (M72II) remained active, with partial loss of enzyme activity, while the enzyme activities of other mutants with alterations at four domains located around amino acid residues 729 to 730 (M73HN), 804 to 807 (M80 and DE80), 910 to 913 (M91 and DE91), and 962 to 964 (M96 and DE96) were abolished. DNA template and triphosphate binding assays indicated that the mutation at 804 to 807 (conserved region III) lost the ability to bind DNA template, and four mutants, M73HN (within conserved region II), M80 (in region III), M91 (in region I), and M96 (around region V [962 to 964; amino acid sequence KKR]), failed to bind deoxyribonucleoside triphosphate. These data suggest that conserved region III is essential for DNA template binding, while residues between conserved region II and V (725 to 964) are involved in triphosphate binding.
Ye
L B
LB
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295.
Huang
E S
ES
D001483:Base Sequence; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000201:enzymology / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015183:Restriction Mapping; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D015678:Viral Structural Proteins; D004259:DNA-Directed DNA Polymerase