An essential arginine residue for initiation of protein-primed DNA replication.
A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins). We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources. The location of this sequence motif is also similar among the different terminal-proteins. To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system. The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants. Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein. Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein. Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP. On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP.
Hsieh
J C
JC
Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724.
Yoo
S K
SK
Ito
J
J
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D003090:Coliphages / Q000235:genetics*; D004261:DNA Replication; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics
C054298:DNA terminal protein, Enterobacteria phage PRD1; D003848:Deoxyguanine Nucleotides; D014764:Viral Proteins; D015678:Viral Structural Proteins; C007257:2'-deoxyguanosine 5'-phosphate; D001120:Arginine