Ah. Rosenberg et al., SELECTION, IDENTIFICATION, AND GENETIC-ANALYSIS OF RANDOM MUTANTS IN THE CLONED PRIMASE HELICASE GENE OF BACTERIOPHAGE-T7/, The Journal of biological chemistry, 271(43), 1996, pp. 26819-26824
T7 gene 4 specifies two overlapping proteins 4A, a 566-amino acid prim
ase/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 leucin
e codon replacing the 4B initiation codon, specifies a single 566-amin
o acid protein that can provide the primase and helicase functions req
uired for normal T7 growth, We selected N-methyl-N'-nitro-N-nitrosogua
nidine mutants in the cloned 4A' gene that no longer support the growt
h of a phage that completely lacks gene 4, Genetic mapping of the 76 m
utations found them to be distributed throughout the protein, includin
g 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 b
ut five of the mutants lacked helicase function but retained primase f
unction, The other five, which lacked both functions, all made short p
roteins, including one missing only 60 amino acids. No mutations lacke
d only primase function, and none mapped within the first 105 amino ac
ids, which includes the 63-amino acid region unique to 4A that contain
s elements required to recognize primase sites, Forty-six mutations we
re sequenced and included 27 missense mutations affecting 25 amino aci
ds, Many mutations in the N-terminal half of the protein affected its
solubility in cell extracts. Mutations in the C-terminal half clustere
d in or near five helicase consensus sequences, Biochemical analysis o
f nine of the mutant proteins is described in the accompanying paper (
Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W., and
Patel, S. S. (1996) J. Biol. Chem. 271, 26825-26834).