Selection, Identification, and Genetic Analysis of Random Mutants in the Cloned Primase/Helicase Gene of Bacteriophage T7

Abstract

T7 gene 4 specifies two overlapping proteins 4A, a 566-amino acid primase/helicase, and 4B, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. The 4A gene, which has a leucine codon replacing the 4B initiation codon, specifies a single 566-amino acid protein that can provide the primase and helicase functions required for normal T7 growth. We selected N-methyl-N-nitro-N-nitrosoguanidine mutants in the cloned 4A gene that no longer support the growth of a phage that completely lacks gene 4. Genetic mapping of the 76 mutations found them to be distributed throughout the protein, including both the N-terminal and C-terminal halves of the molecule thought to represent primase and helicase domains, respectively. Complementation tests with partially and completely defective phage showed that all but five of the mutants lacked helicase function but retained primase function. The other five, which lacked both functions, all made short proteins, including one missing only 60 amino acids. No mutations lacked only primase function, and none mapped within the first 105 amino acids, which includes the 63-amino acid region unique to 4A that contains elements required to recognize primase sites. Forty-six mutations were sequenced and included 27 missense mutations affecting 25 amino acids. Many mutations in the N-terminal half of the protein affected its solubility in cell extracts. Mutations in the C-terminal half clustered in or near five helicase consensus sequences. Biochemical analysis of nine of the mutant proteins is described in the accompanying paper (Washington, M. T.,

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template

“…The mixture of E. coli cells containing the plasmid library was infected with T7 phage ⌬4-1 lacking T7 nucleotides 11,563-11,806 (24). The presence of the T7 nucleotide 11,333-11,950 in the plasmid allows for homologous recombination with the phage genome to produce phages containing various codons at the three positions in the ZBD.…”

Essential Amino Acid Residues in the Single-stranded DNA-binding Protein of Bacteriophage T7

“…To begin mapping these domains, we developed a screen for lethal mutations in gene 2.5. A similar screen was successfully used to identify lethal mutants of the T7 helicase/primase (29). Presumably, mutations that are lethal will occur in regions critical to wt gene 2.5 protein functions or proper folding.…”

The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

“…Furthermore, it is not known if the linker region via its contact with adjacent subunits assists in coordinating helicase and primase activities at the replication fork. One study (15) involving a random mutagenesis of the entire T7 gene 4 protein did find that substitutions of residues within this region such as A257T, A257V, and G258D give rise to proteins that cannot support T7 phage lacking gene 4. One of these altered proteins, a gene 4 protein with the substitution of threonine for alanine at position 257 (gp4-A257T), had reduced dTTPase activity and no DNA unwinding activity.…”