Linked spontaneous CG----TA mutations at CpG sites in the gene for protein kinase regulatory subunit.

Abstract

are thought to result from hydrolytic deamination of 5-methylcytosine residues in these sites. The gene for regulatory subunit of murine cyclic AMP-dependent protein kinase has two closely linked CpG sites, one of which is a strong hotspot for spontaneous CG-+TA mutations leading to cyclic AMP resistance in S49 mouse lymphoma cells. About 5% of mutants with a spontaneous mutation at this CpG site had also acquired a second CG-oTA mutation at the nearby CpG site. The two mutations were always at first positions of the Arg codons in which they occurred, and they were always together in a single regulatory subunit allele. Their linked appearance could be attributed to neither the selection conditions nor the preexistence of one mutation in the target cells. The high frequency of these double mutants suggests that their lesions result not from hydrolytic deamination but rather from an endogenous enzymatic mechanism.Spontaneous point mutations are thought to arise from errors in replicative or repair synthesis of DNA, the chemical effects of background ionizing radiation, and/or the chemical instability of normal or modified DNA bases (6, 18). Their low frequency at any one allele has suggested that the flux of DNA-damaging events is low and distributed throughout the genome. Transition mutations at CpG dinucleotide sequences account for about 25 to 40% of known point mutations leading to human genetic disorders or cancer and are thought to arise by spontaneous hydrolytic deamination of 5-methylcytosine (5-Me-C) residues, which occur most frequently at CpG sites (3, 7, 13). Although it is impossible to know a priori whether these human mutations were spontaneous or induced by exposure to mutagens, support for the deamination pathway comes from a recent report showing that CpG hotspots for mutations in the low-density lipoprotein receptor and the p53 tumor suppressor genes are indeed methylated in vivo (19).Mutants of the cyclic AMP (cAMP)-sensitive S49 mouse lymphoma cell line selected for resistance to cAMP analogs have provided abundant material for study of missense mutations in a mammalian gene. The most common mutants have lesions in the regulatory (R) or cAMP-binding subunit of cAMP-dependent protein kinase that increase apparent constants (Kas) of the enzyme for cAMP-dependent activation (16,23). In a recent study of sequence changes underlying such lesions in S49 sublines hemizygous for expression of mutant R subunits with altered charge, we found 8 distinct single-base-change mutations clustered, for the most part, in regions identified with the two cAMP-binding sites of R subunit, sites A and B (22); subsequently, we have identified 14 additional point mutations associated with Ka phenotypes in hemizygous or heterozygous mutant cells (10). Among spontaneous isolates, the most frequent mutation was a CG-+TA transition in the site B region causing substitution of Trp for Arg-334. Among mutants heterozygous for expression of mutant R subunits, we found several mutageninduced isolates that had this Trp-334 muta...

Polymorphisms and Genomic Organization of Repetitive DNA from Centromeric Regions of Arabidopsis Chromosomes

“…Active processes of C → T transitional mutagenesis have been reported for fungi (Selker et al, 1993) and mammals (Steinberg and Gorman, 1992), which may be mediated by chromosome pairing or through cytosine methylation. In Neurospora, Centola and Carbon (1994) examined the centromere-specific repetitive DNA sequences and considered that the high sequence divergence, high AT content (65%), and predominantly transitional differences between sequences suggested operation of the repeat-induced point mutation (RIP) system (Selker et al, 1993).…”

Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RIα) Subunit of Cyclic AMP-dependent Protein Kinase

“…Plasmids, Bacteria, and Expression of Kinase Subunits-Full-length nonfusion constructs for bacterial expression of murine C␣ and RI␣ subunits were made in the T7 promoter-containing vector pET-8c (or, for the R subunit plasmid, a variant lacking an EcoRI restriction site) as described previously (20,21). Glu-200, Ile-210, Ser-210, Thr-210, and Asp-324 mutations were introduced into the wild-type R subunit plasmid by cassette replacement using polymerase chain reaction-amplified cDNAs from S49 cell mutants (19).…”

Frequent p53 and H-ras Mutations in Benzene- and Ethylene Oxide-Induced Mammary Gland Carcinomas from B6C3F1 Mice

“…Our study showed that C to T transitions were particularly common p53 mutations in spontaneous mammary carcinomas. One mechanism for the formation of these mutations is deamination of 5-methylcytosine (Steinberg and Gorman, 1992). The prevalence of this mutation may be related to the presence of a number of methylated CpG sites within the p53 gene.…”