Modular organization of T4 DNA polymerase. Evidence from phylogenetics.
We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory ("dominant lethal") to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated froms of gp43 that, although inactive for replication, can still exhibit biological specificity.
Wang
C C
CC
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70115, USA.
Yeh
L S
LS
Karam
J D
JD
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D005814:Genes, Viral; D014644:Genetic Variation; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D004279:DNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase