Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.
High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.
Belouchi
A
A
Institut du cancer de Montréal, Centre de Recherche Louis-Charles Simard, Montréal, Canada.
Ouimet
M
M
Dion
P
P
Gaudreault
N
N
Bradley
W E
WE
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair; D005020:Ethyl Methanesulfonate / Q000737:chemistry; D005091:Exons; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009153:Mutagens / Q000737:chemistry; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D013329:Structure-Activity Relationship
D009153:Mutagens; D005020:Ethyl Methanesulfonate; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000228:Adenine Phosphoribosyltransferase