MUTATIONS IN ESCHERICHIA-COLI DNAA WHICH SUPPRESS A DNAX(TS) POLYMERIZATION MUTATION AND ARE DOMINANT WHEN LOCATED IN THE CHROMOSOMAL ALLELE AND RECESSIVE ON PLASMIDS

Extragenic suppressor mutations which had the ability to suppress a dn aX2016(Ts) DNA polymerization defect and which concomitantly caused co ld sensitivity have been characterized within the dnaA initiation gene , When these alleles (designated Cs, Sr) were moved into dndX(+) strai ns, the new mutants became cold sensitive and phenotypically were init iation defective at 20 degrees C (J, R, Walker, J, A. Ramsey, and W, G , Haldenwang, Proc, Natl. Acad, Sci, USA 79:3340-3344, 1982), Detailed localization by marker rescue and DNA sequencing are reported here, O ne mutation changed codon 213 from Ala to Asp, the second changed Arg- 432 to Leu, and the third changed codon 435 from Thr to Lys, It is str iking that two of the three spontaneous mutations occurred in codons 4 32 and 435; these codons are within a very highly conserved, 12-residu e region (K, Skarstad and E, Boye, Biochim, Biophys, Acta 1217:111-130 , 1994; W, Messer and C, Weigel, submitted for publication) which must be critical for one of the DnaA activities, The dominance of wild-typ e and mutant alleles in both initiation and suppression activities was studied, First, in initiation function, the wild-type allele was domi nant over the Cs, Sr alleles, and this dominance was independent of lo cation. That is, the dnaA(+) allele restored growth to dnaA(Cs, Sr) st rains at 20 degrees C independently of which allele was present on the plasmid, The dnaA(Cs, Sr) alleles provided initiator function at 39 d egrees C and were dominant in a dnaA(Ts) host at that temperature, On the other hand, suppression was dominant when the suppressor allele wa s chromosomal but recessive when it was plasmid borne. Furthermore, su ppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele, These data sugges t that the suppressor allele must be integrated into the chromosome, p erhaps at the normal dnaA location, Suppression by dnaA(Cs, Sr) did no t require initiation at oriC; it was observed in strains deleted of or iC and which initiated at an integrated plasmid origin.