Vol. 12, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 767-772 0270-7306/92/020767-06$02.00/0 Copyright © 1992, American Society for Microbiology
Linked Spontaneous CG->TA Mutations at CpG Sites in the Gene for Protein Kinase Regulatory Subunit ROBERT A. STEINBERG* AND KAREN B. GORMAN Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received 11 March 1991/Accepted 19 November 1991
CG-*TA transitions at CpG sequences account for many human point mutations and 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. tion with one or two closely linked additional mutations (10). An additional mutation found in two such isolates was a CG-*TA transition in the codon for Arg-332 causing its replacement with Cys. These two mutations at Arg codons caused a diagnostic two-charge-unit acidic shift in the mutant R subunits that could be detected easily by twodimensional gel electrophoresis (16). Here we report the presence of these two Arg codon lesions in a number of spontaneous cAMP-resistant isolates and provide evidence that both lesions arose spontaneously and simultaneously. The high frequency with which these lesions are found to be linked in spontaneous mutants suggests that they result from a high flux of mutagenic activity localized to a very small genomic target region. It is clearly inconsistent with their production by spontaneous hydrolytic deamination.
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 mutation in combina*
MATERIALS AND METHODS Cell culture and mutant isolation. Wild-type and mutant isolates of S49 subline U36 were grown in suspension culture in Dulbecco's modified Eagle's medium containing glucose (3 g/liter), sodium bicarbonate (2.24 g/liter), and 10o heatinactivated horse serum as described previously (16). For cloning, medium was solidified by the addition of 0.3% SeaPlaque agarose (FMC); where appropriate, the cAMP analog N6,02-dibutyryl-cAMP (Bt2cAMP) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) was included at a final concentration of 0.5 or 0.05 mM, respectively (16, 23). For mutant isolations, populations of 0.6 x 106 to 2.0 x 106 cells were grown from single-cell-derived colonies of subline U36 (see Table 2, footnote a) and the entire populations were plated in the presence of cAMP analogs. Overall mutation rates were estimated from the distributions of numbers of analog-resistant colonies per population by using published tables (14) after correction for control cloning efficiencies. Single isolates from each population containing mutants were grown up under nonselective conditions and analyzed for the presence or absence of site B mutations by DNA sequence analysis as described below. To subclone cells or to determine cloning efficiencies in the presence or absence of cAMP analogs, about 300 to 1,000
Corresponding author. 767
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cells were plated per 60-mm dish (varying with subline and selective conditions to yield 200 to 400 colonies per dish). The diluted cell populations used for plating were counted with a Coulter electronic particle counter to determine the input cell numbers. Wild-type cloning efficiencies on nonselective dishes were generally greater than 0.80. Sequence analysis of mutant cDNAs. Poly(A)-containing RNAs were isolated from postnuclear supernatants of cell extracts by oligo(dT)-cellulose chromatography and reverse transcribed into cDNA as described elsewhere (9, 22). cAMP-binding site B regions of type I R subunit were then amplified by polymerase chain reaction (PCR), using PST and 3PR primers as described previously (22). The amplified DNA fragments were purified by electrophoresis in gels of NuSieve-GTG agarose and sequenced in both directions, using PST and MLU primers (9, 22). To determine whether double mutants had the two mutations in the same or different alleles, a somewhat larger interval was amplified by using BCL and 3PR primers, and a site B fragment cut from the amplified DNA with restriction endonucleases EcoRI and MluI was subcloned into the M13 bacteriophage vector M13um2l (IBI) as described previously (22). Five to six recombinant phage plaques were isolated from each preparation and sequenced with a Sequenase II kit (U.S. Biochemical) as described elsewhere (22) but using the internal PST positive-strand primer. Two-dimensional gel analysis of mutant R subunits. Mutant cells were labeled with [35S]methionine, extracted, and purified by cAMP-affinity chromatography; the purified R subunits were then resolved by high-resolution two-dimensional gel electrophoresis as described previously (16). Patterns from well-characterized sublines (16) were used to calibrate gel patterns for one- and two-charge-unit shifts in R-subunit positions. Statistical analyses. The significance of finding no double mutants among Bt2cAMP-selected subclones of a Trp-334 mutant was analyzed by application of an algorithm for computing exact significance levels in r x c contingency tables as described previously (16) but using Microsoft Fortran to compile the program and an AT&T PC6300 computer to run it. Expression and characterization of mutant R subunits with a Cys-332, Gln-334, Leu-334, or Trp-334 mutation. The coding sequence for murine type I R subunit was introduced from cDNA plasmids into a pET-8c expression vector (24) from which the EcoRI restriction site had been removed (by restriction, filling, and religation); use of NcoI restriction sites in the plasmid and at the 5' end of the gene ensured expression of a full-length, nonfusion protein product under T7 bacteriophage RNA polymerase control. Mutations were introduced into the wild-type plasmid by cassette replacement using EcoRI and MluI restriction sites. Leu-334 and Trp-334 mutations were obtained from PCR-amplified mutant S49 cell cDNAs; Cys-332 and Gln-334 mutations were generated by site-directed mutagenesis using a PCR-based procedure (17). R subunits were induced to levels of about 1 to 4% of cell protein in Escherichia coli BL21(DE3)pLysS (24), and postribosomal supernatant fractions or partially purified R subunits were reconstituted with purified recombinant murine catalytic (C) subunit for assays of kinase activation. Concentrations of R and C subunits were about 1.5 ,uM and 15 nM, respectively, in reconstitutions containing 10 mM 2-[N-morpholino]ethanesulfonic acid (pH 6.5), 0.1 mM EDTA, 2 mM dithiothreitol, 1 mM magnesium sulfate, 50 mM sodium chloride, 1 mg of bovine serum albumin per ml, and 0.1 mM magnesium-ATP. After incuba-
MOL. CELL. BIOL.
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FIG. 1. Analysis of amplified cDNA sequences from R-subunit site B regions of spontaneous single (Trp-334) and double (Cys-332, Trp-334) mutants. Site B regions were amplified from cDNA copies of poly(A)-containing RNAs by using PCR, and the PCR products were purified and sequenced as described in Materials and Methods, using the negative-strand MLU PCR primer. RNAs were from control (a), singly mutant (b), or doubly mutant (c) cells. A, C, G, and T designate dideoxynucleotides in termination reactions; arrowheads indicate wild-type and mutant versions of mutated residues. The wild-type sequence shown is TGGCAGCCCG*AGGACG* ATTCATCAGCA, with asterisks indicating the mutated residues.
tion for 2 h in ice, mixtures were diluted and assayed for protein kinase activity as described previously (22); in all cases, more than 85% of the C subunit was reconstituted into complexes that were inactive in the absence of cAMP. RESULTS Identification of spontaneous Cys-332, Trp-334 double mutants. Mutants were analyzed for the presence of mutations at Arg-332 and Arg-334 codons by amplifying the site B region from R-subunit cDNA and sequencing the resulting DNA products. Figure 1 shows representative sequencing gel patterns for site B regions from wild-type and mutant S49 cell material; since the mutants tested were heterozygous for expression of mutant and wild-type alleles, the mutant patterns showed evidence for both G and A at the mutated positions in these negative-strand sequences. In a previously described collection of 25 independent spontaneous mutants selected for resistance to growth in Bt2cAMP, 12 had chargeshift lesions in R subunit that mapped to cAMP-binding site B; 8 of these had mutant R subunits with a one-charge-unit acidic shift, and 4 (of which 1 was subsequently lost) had mutant R subunits with a two-charge-unit acidic shift (16). When subjected to sequence analysis, the 11 surviving isolates from this group were all found to carry the Trp-334 mutation; those with the two-charge-unit acidic shift in R subunit had the Cys-332 mutation as well. Double mutants thus accounted for about 33% of mutants with the Trp-334 mutation or 16% of all independent spontaneous mutants in this group. The appearance of double mutants cannot be attributed to selective advantage or to mutagenic selection conditions. Although the Trp-334 mutation was found in four of seven spontaneous Ka mutants of an S49 subline hemizygous for R-subunit expression, no double mutants were found in this collection (22, 23). Since the hemizygous mutants were selected by using CPT-cAMP in place of Bt2cAMP, it
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TABLE 1. Cloning efficiencies of single (Trp-334) and double (Cys-332, Trp-334) mutants in the presence and absence of cAMP analogsa Cloning efficiency Subline
Expt
Control
+Bt2cAMP
+CPT-cAMP
Single mutants
U36.B7.R2 U36.B7.R2 U36.BT2 U36.CPT3 Double mutants U36.B8.R1 U36.B8.R1 U36.B2.R1 U36.CPT2
1 2 3 3
0.47 0.87 0.90 0.76
± 0.26 ± 0.04 ± 0.11 ± 0.10
1 2 3 3
0.41 0.63 0.85 0.90
± ± ± ±
0.16 0.17 0.07 0.24
0.11 0.23 0.43 0.12
± ± ± ±
0.06 0.05 0.11 0.06
0.28 ± 0.17 0.54 + 0.12 0.79 ± 0.10 0.95 ± 0.15
0.24 0.21 0.50 0.29
± + ± ±
0.14 0.08 0.04 0.06
0.42 0.79 0.90 0.79
0.03 0.10 0.15 0.11
± ± ± ±
a Single- or double-mutant isolates of S49 subline U36 were subcloned in agarose-containing medium with no additions (control), CPT-cAMP, or Bt2cAMP as described in Materials and Methods. For experiment 1, triplicate samples were plated from two different dilutions to give about 300 or 1,000 cells per 60-mm dish. For experiments 2 and 3, six replicate samples of about 300 to 1,000 cells were plated per dish (number varied with subline and selective conditions to yield about 200 to 400 colonies per dish). Cloning efficiencies (with 95% confidence limits) were calculated by dividing the mean number of colonies by the number of cells plated per dish; for experiment 1, cloning efficiencies were averaged for the two input concentrations.
seemed possible that the dibutyryl analog might have favored or even promoted appearance of the double mutants. Table 1 shows results from three experiments comparing relative cloning efficiencies of single (Trp-334) or double (Cys-332, Trp-334) mutants in the presence (or absence) of these two selective agents. For the most part, the mutants cloned as well in the presence of CPT-cAMP as in its absence. Cloning efficiencies in Bt2cAMP were lower and more variable both between experiments and among strains carrying the same mutation(s). In Bt2cAMP there was a trend favoring recovery of double over single mutant clones, but the differences were small. These experiments suggested that selection with CPT-cAMP would result in both higher mutant recoveries and less bias between single and double mutants. The very high frequency of double mutants among spontaneous Bt2cAMP-resistant mutants made us worry that the analog might have contributed to the mutagenic process. Several lines of evidence suggest that the Cys-332 mutation confers little or no resistance to cAMP analogs: (i) protein kinase from double mutants has activation parameters indistinguishable from those of kinase from single Trp-334 mutants (10), (ii) the second mutation provides little if any cloning advantage over the Trp-334 mutation itself (Table 1), and (iii) kinase reconstituted from recombinant R subunit with the Cys-332 mutation has an activation constant only slightly higher than that for kinase reconstituted with wildtype R subunit (see Fig. 3). Wild-type and Cys-332 mutants, then, would be expected to die shortly after exposure to selective agents. A Trp-334 mutant, on the other hand, would survive treatment with Bt2cAMP and, if the analog were mutagenic, could acquire a second mutation during clonal outgrowth. To test for this possibility, a Trp-334 mutant was subcloned in agarose under Bt2cAMP selection conditions; 45 independent isolates were characterized by either PCR and DNA sequence analysis of the R-subunit site B region (12 isolates) or two-dimensional gel electrophoresis of radiolabeled R subunits (33 isolates). The subclones were all indistinguishable from the parental mutant; none had acquired the Cys-332 mutation (data not shown). This experiment showed not only that Bt2cAMP does not promote mutations at the Arg-332 codon but also that random occurrence of the Cys-332 mutation is relatively infrequent (less than 0.025 per cell per generation). Single (Trp-334) and double (Cys-332, Trp-334) mutants arise randomly and at high frequency. To determine the true
frequencies of spontaneous single and double mutants, we undertook several additional mutant isolation experiments as described in Materials and Methods and Table 2, footnote a. Rates for appearance of CPT-CAMP- and Bt2cAMPresistant mutants were, respectively, about 2.7 x 10-6 and 3.6 x 1O-7 per cell per generation. That the mutants appeared randomly in advance of selection was supported by both the presence of null populations and large variation in numbers of mutants among the different populations (data not shown). Table 2 summarizes sequence data for the 69 new spontaneous mutants isolated in these experiments. Singe mutants containing the Trp-334 mutation were frequent with both selective agents; they accounted for 75% of the CPT-cAMP-resistant isolates, suggesting the extraordinary mutation frequency of 2 x 10-6 per cell per generation at a single C G base pair. Two Cys-332, Trp-334 double mutants were found among the CPT-cAMP-resistant clones, and one was found among the Bt2cAMP-resistant clones. Although double mutants were less frequent than in the initial set of Bt2cAMP-resistant mutants characterized, they -
TABLE 2. Numbers of appearances of Trp-334 single and Cys-332, Trp-334 double mutations in independent spontaneous mutants resistant to cAMP analogsa Mutation
Selective agent
Bt2cAMP
CPT-cAMP
Expt
1 2 1 2
Trp-334 Single 1 8 9 30
Total
Cys-332, Trp-334
Other'
1 0 1 1
1 6 7 4
Double
3 14 17 35
a For the first experiment, freshly cloned populations of S49 subline U36 were grown to sizes of about 6 x 10- and recloned in the presence of either CPT-cAMP or Bt2cAMP as described in Materials and Methods. From 30 populations subjected to each selection, 17 yielded viable CPT-cAMP-resistant mutants and 3 yielded viable Bt2cAMP-resistant mutants. For the second experiment, 42 freshly cloned populations of subline U36 were divided in two after reaching a size of about 10' cells; the subpopulations were grown to sizes of about 106 and 2 x 106, respectively, for selection with CPT-cAMP or Bt2cAMP. Single isolates from each population with viable mutant clones were analyzed for mutations in cAMP-binding site B as described in Materials and Methods. b Includes isolates with a variety of other mutations in R subunit as well as those with no apparent R-subunit mutation (10).
770
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STEINBERG AND GORMAN
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4: FIG. 2. Sequence analysis of subcloned amplified cDNA from a heterozygous double mutant. R-subunit site B DNA was amplified from mutant cDNA and subcloned into an M13 bacteriophage vector; recombinant phage plaques were isolated and sequenced as described in Materials and Methods, using the internal positivestrand PST primer. Representative sequences shown are from an isolate with wild-type sequence (a) and an isolate with both Cys-332 and Trp-334 mutations (b). The wild-type sequence shown is TGCTGATGAATC*GTCCTC*GGGCTGCCACTGTGGT, with asterisks indicating mutated residues as before. Lanes are labeled as in
Fig. 1.
still accounted for more than 5% of all mutants that contained the Trp-334 mutation. (The higher proportion of double mutants in the initial collection probably reflects a biased sampling procedure in which multiple mutants were analyzed from a number of independent populations and only those from each population that were demonstrably different were retained and counted [16].) As could be predicted from the subcloning experiments of Table 1, selection with CPT-cAMP in place of Bt2cAMP resulted in both a higher apparent mutation frequency and a slightly lower yield of double mutants. The Cys-332 and Trp-334 mutations in double mutants are always physically linked. For double mutants from the set of Bt2cAMP-resistant mutants isolated previously, the twocharge-unit acidic shifts in mutant R subunits provided strong evidence for the presence of both mutations in the same R subunit allele (16). Fo'r the three new double mutants, this linkage was confirmed by subcloning PCR products into an M13 bacteriophage vector and sequencing recombinant phage from a number of individual plaques. Representative (positive-strand) sequence patterns are shown in Fig. 2. Each mutant gave recombinant phage that carried either the wild-type or the doubly mutated R subunit sequence; no phage carried only a single mutation. Cys-332 and Gln-334 mutations have little effect on kinase activation. To assess the expected phenotypic effects of either a Cys-332 mutation alone or a second-position transition mutation in the Arg-334 codon (--*Gln), site-directed mutagenesis was used to introduce these mutations into an R-subunit expre'ssion plasmid. The recombinant proteins were reconstituted with C subunit and assayed for cAMPdependent kinase activity. Figure 3 shows the resulting activation curves and, for comparison, activation curves for enzyme reconstituted with either wild-type R subunits or R subunits with a Leu-334 or Trp-334 mutation (both of which have given selectable phenotypes in S49 cells). In contrast to the Leu-334 and Trp-334 mutations, which shifted apparent
IL
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IcAMP], M FIG. 3. cAMP-dependent activation of wild-type and mutant protein kinases reconstituted from recombinant R and C subunits. Wild-type R subunits and R subunits with the mutations indicated were expressed in E. coli and reconstituted with purified recombinant C subunit as described in Materials and Methods. Dilutions of the reconstituted material were assayed for protein kinase activity without or with various concentrations of cAMP (and, for the Trp-334 mutant, with sufficient N6-benzoyl-cAMP to give full activation). Data were normalized by subtracting the cAMP-independent activity and then dividing by the activity at saturating cyclic nucleotide.
Kas for cAMP-dependent kinase activation by about 8- and 65-fold, the Cys-332 and Gln-334 mutations had effects of only about 2-fold or less on kinase activation. DISCUSSION
These results confirm a strong hotspot for spontaneous
CG-*TA transition mutations at the first position of the codon for Arg-334 of type I R subunit. This change was found in almost 70% of 102 independent CPT-cAMP- or Bt2cAMP-resistant mutants analyzed in this and previous studies (16, 22, 23). A second, closely linked CG--TA transition was found in seven of these mutants. The frequency of double mutants among heterozygous CPT-cAMPresistant isolates suggests a rate of occurrence of about 10-7 per cell per generation. If one uses the very high rate of Trp-334 mutations as a standard for single mutations, the expected rate for independent double mutations would be only about 4 x 10-12, or more than 4 orders of magnitude lower. Our failure to observe any spontaneous occurrences of a Cys-332 mutation alone in almost 200 S49 subclones analyzed- in these and other experiments (10, 16, 22, 23) argues against the unlikely possibility that,mutations at the Arg-332 codon occur randomly at a rate sufficient to account for their appearance by independent generation (almost 0.05 per cell per generation). The linked occurrence of the two mutations also is supported by the invariable colocalization of the lesions in the same R-subunit allele and by the absence of the Cys-332 mutation in subclones of a Trp-334 mutant that were isolated in the presence of Bt2cAMP. (Combining the Bt2cAMP-selected mutants from these and previous experiments [16], we found a total of 5 double mutants in 22 demonstrably independent sublines carrying the Trp-334 mutation; because the earlier experiments discounted sub-
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LINKED SPONTANEOUS CG--TA MUTATIONS AT CpG SITES
clones that might have been siblings, the total of independent Trp-334-containing mutants in these experiments could be as high as 60. Depending on the estimate used for independent Trp-334-containing mutants, the finding of no double mutants in 45 Bt2cAMP-selected subclones of a Trp-334 mutant is significant to P values of about 0.006 to 0.077.) The Cys-332 and Trp-334 mutations both occur at CpG dinucleotides, which frequently are methylated in mammalian DNA (7). From studies of the heat-induced deamination of cytosine residues in DNA and of mutagenesis in wild-type and uracil glycosylase-negative E. coli, it has been widely assumed that deamination of 5-Me-C residues at CpG sequences would cause CG-*TA transitions and thereby contribute to spontaneous mutation in mammalian systems (3, 5, 8, 13, 15, 18). Extrapolation from published rates of 5-Me-C deamination in single- and double-stranded DNA at high temperatures and in single-stranded DNA at physiological temperature suggests that deamination of 5-Me-C residues in double-stranded DNA at 37°C occurs at a rate of about 1.2 x 107/h (or about 2 x 10-6/17-h S49 cell doubling time) (2). Because of the probable attenuating effects of protection and/or repair mechanisms in vivo, this rate is almost surely an overestimate of the rate at which such chemical events would cause mutations. The estimated rate of chemical deamination is thus barely sufficient to account for the observed rate of Trp-334 mutations; it is entirely incapable of explaining the high frequency of Cys-332, Trp-334 double mutations. The high frequency of linked mutations suggests either that the two changes are linked mechanistically or that some process generates high local fluxes of mutagenic activity that can simultaneously affect several sites within a restricted target domain. Localty high concentrations of ionization products are unlikely to explain the mutations reported here, since the reactive species generated by irradiation induce transversions more frequently than transitions (11). Oxygen radicals produced in response to copper ions have been shown to induce both single and double transition mutations in a bacterial system (25), but such a mechanism is also inadequate to explain the results reported here; the copperinduced mutations had no apparent specificity for methylated or CpG sites, and the double mutations were all at tandem bases. Error-prone DNA synthesis has been invoked to explain multiple spontaneous mutations found both in shuttle vector systems and in the adenine phosphoribosyltransferase gene of a human carcinoma cell line (12, 21), but in contrast to those described here, the mutations attributed to replication errors were more varied in type and spacing and showed no preference for CpG sites. Because the Cys-332 and Trp-334 mutations are at CpG sites, we favor a mechanism that attributes them to deamination of 5-Me-C residues; since hydrolytic deamination cannot explain their linked occurrence at high frequency, we postulate a deaminating enzyme whose activity is somewhat precessive along the DNA. Enzymatic deamination of 5-Me-C also has been invoked to explain the very high frequency of CG--TA transitions in duplicated genomic sequences in dikaryotic tissue of Neurospora crassa (1). Despite the symmetry of CpG sites in double-stranded DNA, the transitions reported here were all in first positions of Arg codons (i.e., C--T mutations in the nontranscribed DNA strand). This observation raised the possibility that the mutation process was asymmetric with respect to the target DNA strands. The experiment of Fig. 3, however, by showing that the second-position mutation at Arg-334 (--Gln) was ineffective in altering the activity of cAMP-
771
dependent protein kinase, suggested the simpler explanation that the second-position mutation did not confer a selectable phenotype. A second-position transition mutation has been found in the codon for Arg-332 (--His), but only in a mutagen-induced double mutant (with the second mutation at residue 324); neither mutation at Arg-332 had a significant effect on kinase activation (Fig. 3) (4). If a precessive deaminase is responsible for the linkage of transition mutations at CpG sites, the invariable association of Cys-332 rather than His-332 with the Trp-334 mutation suggests that its precession is along a single DNA strand (either in double-stranded DNA or in the single-stranded regions that arise during transcription or replication). Although multiple spontaneous mutations have been described in other systems, we have found only one other reported instance of simultaneous transitions at closely linked CpG sites (20). That hotspots for double mutations like those described here have not been observed in other somatic cell systems might be explained both by the relatively few systems that have been analyzed in depth and by less fortuitous combinations of target sequences and selective conditions in those systems that have been studied. Furthermore, it is possible that the mutational pathway leading to transition mutations at CpG sites is unusually active in S49 cells. ACKNOWLEDGMENTS We thank Robert D. Cauthron and Marina M. Symcox for their substantial assistance in carrying out and preparing recombinant proteins for the reconstitution experiments of Fig. 3. We also thank Robert D. Ivarie and Lawrence A. Loeb for helpful suggestions and encouragement. This work was supported by grant DK37583 from the National Institute of Diabetes and Digestive and Kidney Diseases. REFERENCES 1. Cambareri, E. B., B. C. Jensen, E. Schabtach, and E. U. Selker. 1989. Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571-1575. 2. Cooper, D. N., and M. Krawczak. 1989. Cytosine methylation
3. 4.
5. 6.
7.
8. 9. 10. 11.
12.
and the fate of CpG dinucleotides in vertebrate genomes. Hum. Genet. 83:181-188. Cooper, D. N., and H. Youssoufian. 1988. The CpG dinucleotide and human genetic disease. Hum. Genet. 78:151-155. Correll, L. A., T. A. Woodford, J. D. Corbin, P. L. Mellon, and G. S. McKnight. 1989. Functional characterization of cAMPbinding mutations in type I protein kinase. J. Biol. Chem. 264:16672-16678. Coulondre, C., J. H. Miller, P. J. Farabaugh, and W. Gilbert. 1978. Molecular basis of base substitution hotspots in Escherichia coli. Nature (London) 274:775-780. De Jong, P. J., A. J. Grosovsky, and B. W. Glickman. 1988. Spectrum of spontaneous mutation at the aprt locus of Chinese hamster ovary cells: an analysis at the DNA sequence level. Proc. Natl. Acad. Sci. USA 85:3499-3503. Doerfler, W. 1983. DNA methylation and gene activity. Annu. Rev. Biochem. 52:93-124. Duncan, B. K., and J. H. Miller. 1980. Mutagenic deamination of cytosine residues in DNA. Nature (London) 287:560-561. Gorman, K. B., and R. A. Steinberg. 1989. Simplified method for selective amplification and direct sequencing of cDNAs. BioTechniques 7:326-331. Gorman, K. B., and R. A. Steinberg. Unpublished results. Grosovsky, A. J., J. G. de Boer, P. J. de Jong, E. A. Drobetsky, and B. W. Glickman. 1988. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc. Natl. Acad. Sci. USA 85:185188. Harwood, J., A. Tachibana, and M. Meuth. 1991. Multiple
772
13.
14. 15. 16.
17.
18. 19.
STEINBERG AND GORMAN
dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line. Mol. Cell. Biol. 11:3163-3170. Koeberl, D. D., C. D. K. Bottema, G. Sarkar, R. P. Ketterling, S.-H. Chen, and S. S. Sommer. 1990. Recurrent nonsense mutations at arginine residues cause severe hemophilia B in unrelated hemophiliacs. Hum. Genet. 84:387-390. Lea, D. E., and C. A. Coulson. 1949. The distribution of the numbers of mutants in bacterial populations. J. Genet. 49:264285. Lindahl, T., and B. Nyberg. 1974. Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13: 3405-3410. Murphy, C. S., and R. A. Steinberg. 1985. Hotspots for spontaneous and mutagen-induced lesions in regulatory subunit of cyclic AMP-dependent protein kinase in S49 mouse lymphoma cells. Somatic Cell Mol. Genet. 11:605-615. Nelson, R. M., and G. L. Long. 1989. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal. Biochem. 180:147151. Phear, G., W. Armstrong, and M. Meuth. 1989. Molecular basis of spontaneous mutation at the aprt locus of hamster cells. J. Mol. Biol. 209:577-582. Rideout, W. M., III, G. A. Coetzee, A. F. Olumi, and P. A. Jones. 1990. 5-Methylcytosine as an endogenous mutagen in the
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human LDL receptor and p53 genes. Science 249:1288-1290. 20. Romac, S., P. Leong, H. Sockett, and F. Hutchinson. 1989. DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells. J. Mol. Biol. 209:195-204. 21. Seidman, M. M., A. Bredberg, S. Seetharam, and K. H. Kraemer. 1987. Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity. Proc. Natl. Acad. Sci. USA 84:4944 4948. 22. Steinberg, R. A., K. B. Gorman, D. 0greid, S. 0. D0skeland, and I. T. Weber. 1991. Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells. J. Biol. Chem. 266:3547-3553. 23. Steinberg, R. A., C. S. Murphy, J. L. Russell, and K. B. Gorman. 1987. Cyclic AMP-resistant mutants of S49 mouse lymphoma cells hemizygous for expression of regulatory subunit of type I cyclic AMP-dependent protein kinase. Somatic Cell Mol. Genet. 13:645-659. 24. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60-89. 25. Tkeshelashvili, L. K., T. McBride, K. Spence, and L. A. Loeb. 1991. Mutation spectrum of copper-induced DNA damage. J. Biol. Chem. 266:6401-6406.
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 767-772 0270-7306/92/020767-06$02.00/0 Copyright © 1992, American Society for Microbiology
Linked Spontaneous CG->TA Mutations at CpG Sites in the Gene for Protein Kinase Regulatory Subunit ROBERT A. STEINBERG* AND KAREN B. GORMAN Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received 11 March 1991/Accepted 19 November 1991
CG-*TA transitions at CpG sequences account for many human point mutations and 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. tion with one or two closely linked additional mutations (10). An additional mutation found in two such isolates was a CG-*TA transition in the codon for Arg-332 causing its replacement with Cys. These two mutations at Arg codons caused a diagnostic two-charge-unit acidic shift in the mutant R subunits that could be detected easily by twodimensional gel electrophoresis (16). Here we report the presence of these two Arg codon lesions in a number of spontaneous cAMP-resistant isolates and provide evidence that both lesions arose spontaneously and simultaneously. The high frequency with which these lesions are found to be linked in spontaneous mutants suggests that they result from a high flux of mutagenic activity localized to a very small genomic target region. It is clearly inconsistent with their production by spontaneous hydrolytic deamination.
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 mutation in combina*
MATERIALS AND METHODS Cell culture and mutant isolation. Wild-type and mutant isolates of S49 subline U36 were grown in suspension culture in Dulbecco's modified Eagle's medium containing glucose (3 g/liter), sodium bicarbonate (2.24 g/liter), and 10o heatinactivated horse serum as described previously (16). For cloning, medium was solidified by the addition of 0.3% SeaPlaque agarose (FMC); where appropriate, the cAMP analog N6,02-dibutyryl-cAMP (Bt2cAMP) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) was included at a final concentration of 0.5 or 0.05 mM, respectively (16, 23). For mutant isolations, populations of 0.6 x 106 to 2.0 x 106 cells were grown from single-cell-derived colonies of subline U36 (see Table 2, footnote a) and the entire populations were plated in the presence of cAMP analogs. Overall mutation rates were estimated from the distributions of numbers of analog-resistant colonies per population by using published tables (14) after correction for control cloning efficiencies. Single isolates from each population containing mutants were grown up under nonselective conditions and analyzed for the presence or absence of site B mutations by DNA sequence analysis as described below. To subclone cells or to determine cloning efficiencies in the presence or absence of cAMP analogs, about 300 to 1,000
Corresponding author. 767
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STEINBERG AND GORMAN
cells were plated per 60-mm dish (varying with subline and selective conditions to yield 200 to 400 colonies per dish). The diluted cell populations used for plating were counted with a Coulter electronic particle counter to determine the input cell numbers. Wild-type cloning efficiencies on nonselective dishes were generally greater than 0.80. Sequence analysis of mutant cDNAs. Poly(A)-containing RNAs were isolated from postnuclear supernatants of cell extracts by oligo(dT)-cellulose chromatography and reverse transcribed into cDNA as described elsewhere (9, 22). cAMP-binding site B regions of type I R subunit were then amplified by polymerase chain reaction (PCR), using PST and 3PR primers as described previously (22). The amplified DNA fragments were purified by electrophoresis in gels of NuSieve-GTG agarose and sequenced in both directions, using PST and MLU primers (9, 22). To determine whether double mutants had the two mutations in the same or different alleles, a somewhat larger interval was amplified by using BCL and 3PR primers, and a site B fragment cut from the amplified DNA with restriction endonucleases EcoRI and MluI was subcloned into the M13 bacteriophage vector M13um2l (IBI) as described previously (22). Five to six recombinant phage plaques were isolated from each preparation and sequenced with a Sequenase II kit (U.S. Biochemical) as described elsewhere (22) but using the internal PST positive-strand primer. Two-dimensional gel analysis of mutant R subunits. Mutant cells were labeled with [35S]methionine, extracted, and purified by cAMP-affinity chromatography; the purified R subunits were then resolved by high-resolution two-dimensional gel electrophoresis as described previously (16). Patterns from well-characterized sublines (16) were used to calibrate gel patterns for one- and two-charge-unit shifts in R-subunit positions. Statistical analyses. The significance of finding no double mutants among Bt2cAMP-selected subclones of a Trp-334 mutant was analyzed by application of an algorithm for computing exact significance levels in r x c contingency tables as described previously (16) but using Microsoft Fortran to compile the program and an AT&T PC6300 computer to run it. Expression and characterization of mutant R subunits with a Cys-332, Gln-334, Leu-334, or Trp-334 mutation. The coding sequence for murine type I R subunit was introduced from cDNA plasmids into a pET-8c expression vector (24) from which the EcoRI restriction site had been removed (by restriction, filling, and religation); use of NcoI restriction sites in the plasmid and at the 5' end of the gene ensured expression of a full-length, nonfusion protein product under T7 bacteriophage RNA polymerase control. Mutations were introduced into the wild-type plasmid by cassette replacement using EcoRI and MluI restriction sites. Leu-334 and Trp-334 mutations were obtained from PCR-amplified mutant S49 cell cDNAs; Cys-332 and Gln-334 mutations were generated by site-directed mutagenesis using a PCR-based procedure (17). R subunits were induced to levels of about 1 to 4% of cell protein in Escherichia coli BL21(DE3)pLysS (24), and postribosomal supernatant fractions or partially purified R subunits were reconstituted with purified recombinant murine catalytic (C) subunit for assays of kinase activation. Concentrations of R and C subunits were about 1.5 ,uM and 15 nM, respectively, in reconstitutions containing 10 mM 2-[N-morpholino]ethanesulfonic acid (pH 6.5), 0.1 mM EDTA, 2 mM dithiothreitol, 1 mM magnesium sulfate, 50 mM sodium chloride, 1 mg of bovine serum albumin per ml, and 0.1 mM magnesium-ATP. After incuba-
MOL. CELL. BIOL.
b
a
c
AC GT A CG T AC G T 4-_ q.
....*
i
A.
4--
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auw
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FIG. 1. Analysis of amplified cDNA sequences from R-subunit site B regions of spontaneous single (Trp-334) and double (Cys-332, Trp-334) mutants. Site B regions were amplified from cDNA copies of poly(A)-containing RNAs by using PCR, and the PCR products were purified and sequenced as described in Materials and Methods, using the negative-strand MLU PCR primer. RNAs were from control (a), singly mutant (b), or doubly mutant (c) cells. A, C, G, and T designate dideoxynucleotides in termination reactions; arrowheads indicate wild-type and mutant versions of mutated residues. The wild-type sequence shown is TGGCAGCCCG*AGGACG* ATTCATCAGCA, with asterisks indicating the mutated residues.
tion for 2 h in ice, mixtures were diluted and assayed for protein kinase activity as described previously (22); in all cases, more than 85% of the C subunit was reconstituted into complexes that were inactive in the absence of cAMP. RESULTS Identification of spontaneous Cys-332, Trp-334 double mutants. Mutants were analyzed for the presence of mutations at Arg-332 and Arg-334 codons by amplifying the site B region from R-subunit cDNA and sequencing the resulting DNA products. Figure 1 shows representative sequencing gel patterns for site B regions from wild-type and mutant S49 cell material; since the mutants tested were heterozygous for expression of mutant and wild-type alleles, the mutant patterns showed evidence for both G and A at the mutated positions in these negative-strand sequences. In a previously described collection of 25 independent spontaneous mutants selected for resistance to growth in Bt2cAMP, 12 had chargeshift lesions in R subunit that mapped to cAMP-binding site B; 8 of these had mutant R subunits with a one-charge-unit acidic shift, and 4 (of which 1 was subsequently lost) had mutant R subunits with a two-charge-unit acidic shift (16). When subjected to sequence analysis, the 11 surviving isolates from this group were all found to carry the Trp-334 mutation; those with the two-charge-unit acidic shift in R subunit had the Cys-332 mutation as well. Double mutants thus accounted for about 33% of mutants with the Trp-334 mutation or 16% of all independent spontaneous mutants in this group. The appearance of double mutants cannot be attributed to selective advantage or to mutagenic selection conditions. Although the Trp-334 mutation was found in four of seven spontaneous Ka mutants of an S49 subline hemizygous for R-subunit expression, no double mutants were found in this collection (22, 23). Since the hemizygous mutants were selected by using CPT-cAMP in place of Bt2cAMP, it
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LINKED SPONTANEOUS CG-)TA MUTATIONS AT CpG SITES
VOL. 12, 1992
TABLE 1. Cloning efficiencies of single (Trp-334) and double (Cys-332, Trp-334) mutants in the presence and absence of cAMP analogsa Cloning efficiency Subline
Expt
Control
+Bt2cAMP
+CPT-cAMP
Single mutants
U36.B7.R2 U36.B7.R2 U36.BT2 U36.CPT3 Double mutants U36.B8.R1 U36.B8.R1 U36.B2.R1 U36.CPT2
1 2 3 3
0.47 0.87 0.90 0.76
± 0.26 ± 0.04 ± 0.11 ± 0.10
1 2 3 3
0.41 0.63 0.85 0.90
± ± ± ±
0.16 0.17 0.07 0.24
0.11 0.23 0.43 0.12
± ± ± ±
0.06 0.05 0.11 0.06
0.28 ± 0.17 0.54 + 0.12 0.79 ± 0.10 0.95 ± 0.15
0.24 0.21 0.50 0.29
± + ± ±
0.14 0.08 0.04 0.06
0.42 0.79 0.90 0.79
0.03 0.10 0.15 0.11
± ± ± ±
a Single- or double-mutant isolates of S49 subline U36 were subcloned in agarose-containing medium with no additions (control), CPT-cAMP, or Bt2cAMP as described in Materials and Methods. For experiment 1, triplicate samples were plated from two different dilutions to give about 300 or 1,000 cells per 60-mm dish. For experiments 2 and 3, six replicate samples of about 300 to 1,000 cells were plated per dish (number varied with subline and selective conditions to yield about 200 to 400 colonies per dish). Cloning efficiencies (with 95% confidence limits) were calculated by dividing the mean number of colonies by the number of cells plated per dish; for experiment 1, cloning efficiencies were averaged for the two input concentrations.
seemed possible that the dibutyryl analog might have favored or even promoted appearance of the double mutants. Table 1 shows results from three experiments comparing relative cloning efficiencies of single (Trp-334) or double (Cys-332, Trp-334) mutants in the presence (or absence) of these two selective agents. For the most part, the mutants cloned as well in the presence of CPT-cAMP as in its absence. Cloning efficiencies in Bt2cAMP were lower and more variable both between experiments and among strains carrying the same mutation(s). In Bt2cAMP there was a trend favoring recovery of double over single mutant clones, but the differences were small. These experiments suggested that selection with CPT-cAMP would result in both higher mutant recoveries and less bias between single and double mutants. The very high frequency of double mutants among spontaneous Bt2cAMP-resistant mutants made us worry that the analog might have contributed to the mutagenic process. Several lines of evidence suggest that the Cys-332 mutation confers little or no resistance to cAMP analogs: (i) protein kinase from double mutants has activation parameters indistinguishable from those of kinase from single Trp-334 mutants (10), (ii) the second mutation provides little if any cloning advantage over the Trp-334 mutation itself (Table 1), and (iii) kinase reconstituted from recombinant R subunit with the Cys-332 mutation has an activation constant only slightly higher than that for kinase reconstituted with wildtype R subunit (see Fig. 3). Wild-type and Cys-332 mutants, then, would be expected to die shortly after exposure to selective agents. A Trp-334 mutant, on the other hand, would survive treatment with Bt2cAMP and, if the analog were mutagenic, could acquire a second mutation during clonal outgrowth. To test for this possibility, a Trp-334 mutant was subcloned in agarose under Bt2cAMP selection conditions; 45 independent isolates were characterized by either PCR and DNA sequence analysis of the R-subunit site B region (12 isolates) or two-dimensional gel electrophoresis of radiolabeled R subunits (33 isolates). The subclones were all indistinguishable from the parental mutant; none had acquired the Cys-332 mutation (data not shown). This experiment showed not only that Bt2cAMP does not promote mutations at the Arg-332 codon but also that random occurrence of the Cys-332 mutation is relatively infrequent (less than 0.025 per cell per generation). Single (Trp-334) and double (Cys-332, Trp-334) mutants arise randomly and at high frequency. To determine the true
frequencies of spontaneous single and double mutants, we undertook several additional mutant isolation experiments as described in Materials and Methods and Table 2, footnote a. Rates for appearance of CPT-CAMP- and Bt2cAMPresistant mutants were, respectively, about 2.7 x 10-6 and 3.6 x 1O-7 per cell per generation. That the mutants appeared randomly in advance of selection was supported by both the presence of null populations and large variation in numbers of mutants among the different populations (data not shown). Table 2 summarizes sequence data for the 69 new spontaneous mutants isolated in these experiments. Singe mutants containing the Trp-334 mutation were frequent with both selective agents; they accounted for 75% of the CPT-cAMP-resistant isolates, suggesting the extraordinary mutation frequency of 2 x 10-6 per cell per generation at a single C G base pair. Two Cys-332, Trp-334 double mutants were found among the CPT-cAMP-resistant clones, and one was found among the Bt2cAMP-resistant clones. Although double mutants were less frequent than in the initial set of Bt2cAMP-resistant mutants characterized, they -
TABLE 2. Numbers of appearances of Trp-334 single and Cys-332, Trp-334 double mutations in independent spontaneous mutants resistant to cAMP analogsa Mutation
Selective agent
Bt2cAMP
CPT-cAMP
Expt
1 2 1 2
Trp-334 Single 1 8 9 30
Total
Cys-332, Trp-334
Other'
1 0 1 1
1 6 7 4
Double
3 14 17 35
a For the first experiment, freshly cloned populations of S49 subline U36 were grown to sizes of about 6 x 10- and recloned in the presence of either CPT-cAMP or Bt2cAMP as described in Materials and Methods. From 30 populations subjected to each selection, 17 yielded viable CPT-cAMP-resistant mutants and 3 yielded viable Bt2cAMP-resistant mutants. For the second experiment, 42 freshly cloned populations of subline U36 were divided in two after reaching a size of about 10' cells; the subpopulations were grown to sizes of about 106 and 2 x 106, respectively, for selection with CPT-cAMP or Bt2cAMP. Single isolates from each population with viable mutant clones were analyzed for mutations in cAMP-binding site B as described in Materials and Methods. b Includes isolates with a variety of other mutations in R subunit as well as those with no apparent R-subunit mutation (10).
770
MOL. CELL. BIOL.
STEINBERG AND GORMAN
a A CG T
z 0
b A CG T
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4: FIG. 2. Sequence analysis of subcloned amplified cDNA from a heterozygous double mutant. R-subunit site B DNA was amplified from mutant cDNA and subcloned into an M13 bacteriophage vector; recombinant phage plaques were isolated and sequenced as described in Materials and Methods, using the internal positivestrand PST primer. Representative sequences shown are from an isolate with wild-type sequence (a) and an isolate with both Cys-332 and Trp-334 mutations (b). The wild-type sequence shown is TGCTGATGAATC*GTCCTC*GGGCTGCCACTGTGGT, with asterisks indicating mutated residues as before. Lanes are labeled as in
Fig. 1.
still accounted for more than 5% of all mutants that contained the Trp-334 mutation. (The higher proportion of double mutants in the initial collection probably reflects a biased sampling procedure in which multiple mutants were analyzed from a number of independent populations and only those from each population that were demonstrably different were retained and counted [16].) As could be predicted from the subcloning experiments of Table 1, selection with CPT-cAMP in place of Bt2cAMP resulted in both a higher apparent mutation frequency and a slightly lower yield of double mutants. The Cys-332 and Trp-334 mutations in double mutants are always physically linked. For double mutants from the set of Bt2cAMP-resistant mutants isolated previously, the twocharge-unit acidic shifts in mutant R subunits provided strong evidence for the presence of both mutations in the same R subunit allele (16). Fo'r the three new double mutants, this linkage was confirmed by subcloning PCR products into an M13 bacteriophage vector and sequencing recombinant phage from a number of individual plaques. Representative (positive-strand) sequence patterns are shown in Fig. 2. Each mutant gave recombinant phage that carried either the wild-type or the doubly mutated R subunit sequence; no phage carried only a single mutation. Cys-332 and Gln-334 mutations have little effect on kinase activation. To assess the expected phenotypic effects of either a Cys-332 mutation alone or a second-position transition mutation in the Arg-334 codon (--*Gln), site-directed mutagenesis was used to introduce these mutations into an R-subunit expre'ssion plasmid. The recombinant proteins were reconstituted with C subunit and assayed for cAMPdependent kinase activity. Figure 3 shows the resulting activation curves and, for comparison, activation curves for enzyme reconstituted with either wild-type R subunits or R subunits with a Leu-334 or Trp-334 mutation (both of which have given selectable phenotypes in S49 cells). In contrast to the Leu-334 and Trp-334 mutations, which shifted apparent
IL
lo-,,
107c
P
Mo lo-,
IcAMP], M FIG. 3. cAMP-dependent activation of wild-type and mutant protein kinases reconstituted from recombinant R and C subunits. Wild-type R subunits and R subunits with the mutations indicated were expressed in E. coli and reconstituted with purified recombinant C subunit as described in Materials and Methods. Dilutions of the reconstituted material were assayed for protein kinase activity without or with various concentrations of cAMP (and, for the Trp-334 mutant, with sufficient N6-benzoyl-cAMP to give full activation). Data were normalized by subtracting the cAMP-independent activity and then dividing by the activity at saturating cyclic nucleotide.
Kas for cAMP-dependent kinase activation by about 8- and 65-fold, the Cys-332 and Gln-334 mutations had effects of only about 2-fold or less on kinase activation. DISCUSSION
These results confirm a strong hotspot for spontaneous
CG-*TA transition mutations at the first position of the codon for Arg-334 of type I R subunit. This change was found in almost 70% of 102 independent CPT-cAMP- or Bt2cAMP-resistant mutants analyzed in this and previous studies (16, 22, 23). A second, closely linked CG--TA transition was found in seven of these mutants. The frequency of double mutants among heterozygous CPT-cAMPresistant isolates suggests a rate of occurrence of about 10-7 per cell per generation. If one uses the very high rate of Trp-334 mutations as a standard for single mutations, the expected rate for independent double mutations would be only about 4 x 10-12, or more than 4 orders of magnitude lower. Our failure to observe any spontaneous occurrences of a Cys-332 mutation alone in almost 200 S49 subclones analyzed- in these and other experiments (10, 16, 22, 23) argues against the unlikely possibility that,mutations at the Arg-332 codon occur randomly at a rate sufficient to account for their appearance by independent generation (almost 0.05 per cell per generation). The linked occurrence of the two mutations also is supported by the invariable colocalization of the lesions in the same R-subunit allele and by the absence of the Cys-332 mutation in subclones of a Trp-334 mutant that were isolated in the presence of Bt2cAMP. (Combining the Bt2cAMP-selected mutants from these and previous experiments [16], we found a total of 5 double mutants in 22 demonstrably independent sublines carrying the Trp-334 mutation; because the earlier experiments discounted sub-
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LINKED SPONTANEOUS CG--TA MUTATIONS AT CpG SITES
clones that might have been siblings, the total of independent Trp-334-containing mutants in these experiments could be as high as 60. Depending on the estimate used for independent Trp-334-containing mutants, the finding of no double mutants in 45 Bt2cAMP-selected subclones of a Trp-334 mutant is significant to P values of about 0.006 to 0.077.) The Cys-332 and Trp-334 mutations both occur at CpG dinucleotides, which frequently are methylated in mammalian DNA (7). From studies of the heat-induced deamination of cytosine residues in DNA and of mutagenesis in wild-type and uracil glycosylase-negative E. coli, it has been widely assumed that deamination of 5-Me-C residues at CpG sequences would cause CG-*TA transitions and thereby contribute to spontaneous mutation in mammalian systems (3, 5, 8, 13, 15, 18). Extrapolation from published rates of 5-Me-C deamination in single- and double-stranded DNA at high temperatures and in single-stranded DNA at physiological temperature suggests that deamination of 5-Me-C residues in double-stranded DNA at 37°C occurs at a rate of about 1.2 x 107/h (or about 2 x 10-6/17-h S49 cell doubling time) (2). Because of the probable attenuating effects of protection and/or repair mechanisms in vivo, this rate is almost surely an overestimate of the rate at which such chemical events would cause mutations. The estimated rate of chemical deamination is thus barely sufficient to account for the observed rate of Trp-334 mutations; it is entirely incapable of explaining the high frequency of Cys-332, Trp-334 double mutations. The high frequency of linked mutations suggests either that the two changes are linked mechanistically or that some process generates high local fluxes of mutagenic activity that can simultaneously affect several sites within a restricted target domain. Localty high concentrations of ionization products are unlikely to explain the mutations reported here, since the reactive species generated by irradiation induce transversions more frequently than transitions (11). Oxygen radicals produced in response to copper ions have been shown to induce both single and double transition mutations in a bacterial system (25), but such a mechanism is also inadequate to explain the results reported here; the copperinduced mutations had no apparent specificity for methylated or CpG sites, and the double mutations were all at tandem bases. Error-prone DNA synthesis has been invoked to explain multiple spontaneous mutations found both in shuttle vector systems and in the adenine phosphoribosyltransferase gene of a human carcinoma cell line (12, 21), but in contrast to those described here, the mutations attributed to replication errors were more varied in type and spacing and showed no preference for CpG sites. Because the Cys-332 and Trp-334 mutations are at CpG sites, we favor a mechanism that attributes them to deamination of 5-Me-C residues; since hydrolytic deamination cannot explain their linked occurrence at high frequency, we postulate a deaminating enzyme whose activity is somewhat precessive along the DNA. Enzymatic deamination of 5-Me-C also has been invoked to explain the very high frequency of CG--TA transitions in duplicated genomic sequences in dikaryotic tissue of Neurospora crassa (1). Despite the symmetry of CpG sites in double-stranded DNA, the transitions reported here were all in first positions of Arg codons (i.e., C--T mutations in the nontranscribed DNA strand). This observation raised the possibility that the mutation process was asymmetric with respect to the target DNA strands. The experiment of Fig. 3, however, by showing that the second-position mutation at Arg-334 (--Gln) was ineffective in altering the activity of cAMP-
771
dependent protein kinase, suggested the simpler explanation that the second-position mutation did not confer a selectable phenotype. A second-position transition mutation has been found in the codon for Arg-332 (--His), but only in a mutagen-induced double mutant (with the second mutation at residue 324); neither mutation at Arg-332 had a significant effect on kinase activation (Fig. 3) (4). If a precessive deaminase is responsible for the linkage of transition mutations at CpG sites, the invariable association of Cys-332 rather than His-332 with the Trp-334 mutation suggests that its precession is along a single DNA strand (either in double-stranded DNA or in the single-stranded regions that arise during transcription or replication). Although multiple spontaneous mutations have been described in other systems, we have found only one other reported instance of simultaneous transitions at closely linked CpG sites (20). That hotspots for double mutations like those described here have not been observed in other somatic cell systems might be explained both by the relatively few systems that have been analyzed in depth and by less fortuitous combinations of target sequences and selective conditions in those systems that have been studied. Furthermore, it is possible that the mutational pathway leading to transition mutations at CpG sites is unusually active in S49 cells. ACKNOWLEDGMENTS We thank Robert D. Cauthron and Marina M. Symcox for their substantial assistance in carrying out and preparing recombinant proteins for the reconstitution experiments of Fig. 3. We also thank Robert D. Ivarie and Lawrence A. Loeb for helpful suggestions and encouragement. This work was supported by grant DK37583 from the National Institute of Diabetes and Digestive and Kidney Diseases. REFERENCES 1. Cambareri, E. B., B. C. Jensen, E. Schabtach, and E. U. Selker. 1989. Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571-1575. 2. Cooper, D. N., and M. Krawczak. 1989. Cytosine methylation
3. 4.
5. 6.
7.
8. 9. 10. 11.
12.
and the fate of CpG dinucleotides in vertebrate genomes. Hum. Genet. 83:181-188. Cooper, D. N., and H. Youssoufian. 1988. The CpG dinucleotide and human genetic disease. Hum. Genet. 78:151-155. Correll, L. A., T. A. Woodford, J. D. Corbin, P. L. Mellon, and G. S. McKnight. 1989. Functional characterization of cAMPbinding mutations in type I protein kinase. J. Biol. Chem. 264:16672-16678. Coulondre, C., J. H. Miller, P. J. Farabaugh, and W. Gilbert. 1978. Molecular basis of base substitution hotspots in Escherichia coli. Nature (London) 274:775-780. De Jong, P. J., A. J. Grosovsky, and B. W. Glickman. 1988. Spectrum of spontaneous mutation at the aprt locus of Chinese hamster ovary cells: an analysis at the DNA sequence level. Proc. Natl. Acad. Sci. USA 85:3499-3503. Doerfler, W. 1983. DNA methylation and gene activity. Annu. Rev. Biochem. 52:93-124. Duncan, B. K., and J. H. Miller. 1980. Mutagenic deamination of cytosine residues in DNA. Nature (London) 287:560-561. Gorman, K. B., and R. A. Steinberg. 1989. Simplified method for selective amplification and direct sequencing of cDNAs. BioTechniques 7:326-331. Gorman, K. B., and R. A. Steinberg. Unpublished results. Grosovsky, A. J., J. G. de Boer, P. J. de Jong, E. A. Drobetsky, and B. W. Glickman. 1988. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc. Natl. Acad. Sci. USA 85:185188. Harwood, J., A. Tachibana, and M. Meuth. 1991. Multiple
772
13.
14. 15. 16.
17.
18. 19.
STEINBERG AND GORMAN
dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line. Mol. Cell. Biol. 11:3163-3170. Koeberl, D. D., C. D. K. Bottema, G. Sarkar, R. P. Ketterling, S.-H. Chen, and S. S. Sommer. 1990. Recurrent nonsense mutations at arginine residues cause severe hemophilia B in unrelated hemophiliacs. Hum. Genet. 84:387-390. Lea, D. E., and C. A. Coulson. 1949. The distribution of the numbers of mutants in bacterial populations. J. Genet. 49:264285. Lindahl, T., and B. Nyberg. 1974. Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13: 3405-3410. Murphy, C. S., and R. A. Steinberg. 1985. Hotspots for spontaneous and mutagen-induced lesions in regulatory subunit of cyclic AMP-dependent protein kinase in S49 mouse lymphoma cells. Somatic Cell Mol. Genet. 11:605-615. Nelson, R. M., and G. L. Long. 1989. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal. Biochem. 180:147151. Phear, G., W. Armstrong, and M. Meuth. 1989. Molecular basis of spontaneous mutation at the aprt locus of hamster cells. J. Mol. Biol. 209:577-582. Rideout, W. M., III, G. A. Coetzee, A. F. Olumi, and P. A. Jones. 1990. 5-Methylcytosine as an endogenous mutagen in the
MOL. CELL. BIOL.
human LDL receptor and p53 genes. Science 249:1288-1290. 20. Romac, S., P. Leong, H. Sockett, and F. Hutchinson. 1989. DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells. J. Mol. Biol. 209:195-204. 21. Seidman, M. M., A. Bredberg, S. Seetharam, and K. H. Kraemer. 1987. Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity. Proc. Natl. Acad. Sci. USA 84:4944 4948. 22. Steinberg, R. A., K. B. Gorman, D. 0greid, S. 0. D0skeland, and I. T. Weber. 1991. Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells. J. Biol. Chem. 266:3547-3553. 23. Steinberg, R. A., C. S. Murphy, J. L. Russell, and K. B. Gorman. 1987. Cyclic AMP-resistant mutants of S49 mouse lymphoma cells hemizygous for expression of regulatory subunit of type I cyclic AMP-dependent protein kinase. Somatic Cell Mol. Genet. 13:645-659. 24. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60-89. 25. Tkeshelashvili, L. K., T. McBride, K. Spence, and L. A. Loeb. 1991. Mutation spectrum of copper-induced DNA damage. J. Biol. Chem. 266:6401-6406.
