SELECTION, IDENTIFICATION, AND GENETIC-ANALYSIS OF RANDOM MUTANTS IN THE CLONED PRIMASE HELICASE GENE OF BACTERIOPHAGE-T7/

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).