MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 767-772 Vol. 12, No. 2 0270-7306/92/020767-06$02.00/0 Copyright © 1992, American Society for Microbiology Linked Spontaneous CG->TA at CpG Sites in the Gene for Kinase Regulatory Subunit ROBERT A. STEINBERG* AND KAREN B. GORMAN Department ofBiochemistry 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 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 with a spontaneous 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 tion with one or two closely linked additional mutations (10). errors in replicative or repair synthesis of DNA, the chem- An additional mutation found in two such isolates was a ical effects of background ionizing radiation, and/or the CG-*TA in the codon for Arg-332 causing its chemical instability of normal or modified DNA bases (6, replacement with Cys. These two mutations at Arg codons 18). Their low frequency at any one allele has suggested that caused a diagnostic two-charge-unit acidic shift in the mu- the flux of DNA-damaging events is low and distributed tant R subunits that could be detected easily by two- throughout the genome. Transition mutations at CpG dinu- dimensional gel electrophoresis (16). Here we report the cleotide sequences account for about 25 to 40% of known presence of these two Arg codon lesions in a number of point mutations leading to human genetic disorders or spontaneous cAMP-resistant isolates and provide evidence and are thought to arise by spontaneous hydrolytic deami- that both lesions arose spontaneously and simultaneously. nation of 5-methylcytosine (5-Me-C) residues, which occur The high frequency with which these lesions are found to be most frequently at CpG sites (3, 7, 13). Although it is linked in spontaneous mutants suggests that they result from impossible to know a priori whether these human mutations a high flux of mutagenic activity localized to a very small were spontaneous or induced by exposure to , genomic target region. It is clearly inconsistent with their support for the deamination pathway comes from a recent production by spontaneous hydrolytic deamination. report showing that CpG hotspots for mutations in the low-density lipoprotein receptor and the p53 tumor suppres- sor genes are indeed methylated in vivo (19). MATERIALS AND METHODS Mutants of the cyclic AMP (cAMP)-sensitive S49 mouse Cell culture and isolation. Wild-type and mutant lymphoma cell line selected for resistance to cAMP analogs isolates of S49 subline U36 were grown in suspension culture have provided abundant material for study of missense in Dulbecco's modified Eagle's medium containing glucose mutations in a mammalian gene. The most common mutants (3 g/liter), sodium bicarbonate (2.24 g/liter), and 10o heat- have lesions in the regulatory (R) or cAMP-binding subunit inactivated horse serum as described previously (16). For of cAMP-dependent protein kinase that increase apparent cloning, medium was solidified by the addition of 0.3% constants (Kas) of the for cAMP-dependent activa- SeaPlaque agarose (FMC); where appropriate, the cAMP tion (16, 23). In a recent study of sequence changes under- analog N6,02-dibutyryl-cAMP (Bt2cAMP) or 8-(4-chlo- lying such lesions in S49 sublines hemizygous for expression rophenylthio)-cAMP (CPT-cAMP) was included at a final of mutant R subunits with altered charge, we found 8 distinct concentration of 0.5 or 0.05 mM, respectively (16, 23). single-base-change mutations clustered, for the most part, in For mutant isolations, populations of 0.6 x 106 to 2.0 x regions identified with the two cAMP-binding sites of R 106 cells were grown from single-cell-derived colonies of subunit, sites A and B (22); subsequently, we have identified subline U36 (see Table 2, footnote a) and the entire popula- 14 additional point mutations associated with Ka phenotypes tions were plated in the presence of cAMP analogs. Overall in hemizygous or heterozygous mutant cells (10). Among mutation rates were estimated from the distributions of spontaneous isolates, the most frequent mutation was a numbers of analog-resistant colonies per population by using CG-+TA transition in the site B region causing substitution published tables (14) after correction for control cloning ofTrp for Arg-334. Among mutants heterozygous for expres- efficiencies. Single isolates from each population containing sion of mutant R subunits, we found several - mutants were grown up under nonselective conditions and induced isolates that had this Trp-334 mutation in combina- 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 * Corresponding author. the presence or absence ofcAMP analogs, about 300 to 1,000 767 768 STEINBERG AND GORMAN MOL. CELL. BIOL. cells were plated per 60-mm dish (varying with subline and selective conditions to yield 200 to 400 colonies per dish). a b c The diluted cell populations used for plating were counted AC GT A CG T AC G T with a Coulter electronic particle counter to determine the 4-_ input cell numbers. Wild-type cloning efficiencies on nonse- i A. q. - lective dishes were generally greater than 0.80. ....* 4-- Sequence analysis of mutant cDNAs. Poly(A)-containing *K RNAs were isolated from postnuclear supernatants of cell -4 extracts by oligo(dT)-cellulose chromatography and reverse 4- transcribed into cDNA as described elsewhere (9, 22). auw cAMP-binding site B regions of type I R subunit were then .p amplified by polymerase chain reaction (PCR), using PST 4 and 3PR primers as described previously (22). The amplified

, 01 DNA fragments were purified by electrophoresis in gels of Wm - * ... .s NuSieve-GTG agarose and sequenced in both directions, Wm using PST and MLU primers (9, 22). To determine whether double mutants had the two muta- tions in the same or different alleles, a somewhat larger FIG. 1. Analysis of amplified cDNA sequences from R-subunit site B regions of spontaneous single (Trp-334) and double (Cys-332, interval was amplified by using BCL and 3PR primers, and a Trp-334) mutants. Site B regions were amplified from cDNA copies site B fragment cut from the amplified DNA with restriction of poly(A)-containing RNAs by using PCR, and the PCR products endonucleases EcoRI and MluI was subcloned into the M13 were purified and sequenced as described in Materials and Methods, bacteriophage vector M13um2l (IBI) as described previ- using the negative-strand MLU PCR primer. RNAs were from ously (22). Five to six recombinant phage plaques were control (a), singly mutant (b), or doubly mutant (c) cells. A, C, G, isolated from each preparation and sequenced with a Seque- and T designate dideoxynucleotides in termination reactions; arrow- nase II kit (U.S. Biochemical) as described elsewhere (22) heads indicate wild-type and mutant versions of mutated residues. but using the internal PST positive-strand primer. The wild-type sequence shown is TGGCAGCCCG*AGGACG* Two-dimensional gel analysis of mutant R subunits. Mutant ATTCATCAGCA, with asterisks indicating the mutated residues. cells were labeled with [35S]methionine, extracted, and purified by cAMP-affinity chromatography; the purified R subunits were then resolved by high-resolution two-dimen- tion for 2 h in ice, mixtures were diluted and assayed for sional gel electrophoresis as described previously (16). Pat- protein kinase activity as described previously (22); in all terns from well-characterized sublines (16) were used to cases, more than 85% of the C subunit was reconstituted into calibrate gel patterns for one- and two-charge-unit shifts in complexes that were inactive in the absence of cAMP. R-subunit positions. Statistical analyses. The significance of finding no double RESULTS mutants among Bt2cAMP-selected subclones of a Trp-334 mutant was analyzed by application of an algorithm for Identification of spontaneous Cys-332, Trp-334 double mu- computing exact significance levels in r x c contingency tants. Mutants were analyzed for the presence of mutations tables as described previously (16) but using Microsoft at Arg-332 and Arg-334 codons by amplifying the site B Fortran to compile the program and an AT&T PC6300 region from R-subunit cDNA and sequencing the resulting computer to run it. DNA products. Figure 1 shows representative sequencing Expression and characterization of mutant R subunits with gel patterns for site B regions from wild-type and mutant S49 a Cys-332, Gln-334, Leu-334, or Trp-334 mutation. The cell material; since the mutants tested were heterozygous for coding sequence for murine type I R subunit was introduced expression of mutant and wild-type alleles, the mutant from cDNA plasmids into a pET-8c expression vector (24) patterns showed evidence for both G and A at the mutated from which the EcoRI restriction site had been removed (by positions in these negative-strand sequences. In a previously restriction, filling, and religation); use of NcoI restriction described collection of 25 independent spontaneous mutants sites in the plasmid and at the 5' end of the gene ensured selected for resistance to growth in Bt2cAMP, 12 had charge- expression of a full-length, nonfusion protein product under shift lesions in R subunit that mapped to cAMP-binding site T7 bacteriophage RNA polymerase control. Mutations were B; 8 of these had mutant R subunits with a one-charge-unit introduced into the wild-type plasmid by cassette replace- acidic shift, and 4 (of which 1 was subsequently lost) had ment using EcoRI and MluI restriction sites. Leu-334 and mutant R subunits with a two-charge-unit acidic shift (16). Trp-334 mutations were obtained from PCR-amplified mu- When subjected to sequence analysis, the 11 surviving tant S49 cell cDNAs; Cys-332 and Gln-334 mutations were isolates from this group were all found to carry the Trp-334 generated by site-directed using a PCR-based mutation; those with the two-charge-unit acidic shift in R procedure (17). R subunits were induced to levels of about 1 subunit had the Cys-332 mutation as well. Double mutants to 4% of cell protein in Escherichia coli BL21(DE3)pLysS thus accounted for about 33% of mutants with the Trp-334 (24), and postribosomal supernatant fractions or partially mutation or 16% of all independent spontaneous mutants in purified R subunits were reconstituted with purified recom- this group. binant murine catalytic (C) subunit for assays of kinase The appearance of double mutants cannot be attributed to activation. Concentrations of R and C subunits were about selective advantage or to mutagenic selection conditions. Al- 1.5 ,uM and 15 nM, respectively, in reconstitutions contain- though the Trp-334 mutation was found in four of seven ing 10 mM 2-[N-morpholino]ethanesulfonic acid (pH 6.5), spontaneous Ka mutants of an S49 subline hemizygous for 0.1 mM EDTA, 2 mM dithiothreitol, 1 mM magnesium R-subunit expression, no double mutants were found in this sulfate, 50 mM sodium chloride, 1 mg of bovine serum collection (22, 23). Since the hemizygous mutants were albumin per ml, and 0.1 mM magnesium-ATP. After incuba- selected by using CPT-cAMP in place of Bt2cAMP, it VOL. 12, 1992 LINKED SPONTANEOUS CG-)TA MUTATIONS AT CpG SITES 769

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 +CPT-cAMP +Bt2cAMP Single mutants U36.B7.R2 1 0.47 ± 0.26 0.42 ± 0.16 0.11 ± 0.06 U36.B7.R2 2 0.87 ± 0.04 0.79 ± 0.17 0.23 ± 0.05 U36.BT2 3 0.90 ± 0.11 0.90 ± 0.07 0.43 ± 0.11 U36.CPT3 3 0.76 ± 0.10 0.79 ± 0.24 0.12 ± 0.06 Double mutants U36.B8.R1 1 0.41 ± 0.03 0.28 ± 0.17 0.24 ± 0.14 U36.B8.R1 2 0.63 ± 0.10 0.54 + 0.12 0.21 + 0.08 U36.B2.R1 3 0.85 ± 0.15 0.79 ± 0.10 0.50 ± 0.04 U36.CPT2 3 0.90 ± 0.11 0.95 ± 0.15 0.29 ± 0.06 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 fa- frequencies of spontaneous single and double mutants, we vored or even promoted appearance of the double mutants. undertook several additional mutant isolation experiments Table 1 shows results from three experiments comparing as described in Materials and Methods and Table 2, footnote relative cloning efficiencies of single (Trp-334) or double a. Rates for appearance of CPT-CAMP- and Bt2cAMP- (Cys-332, Trp-334) mutants in the presence (or absence) of resistant mutants were, respectively, about 2.7 x 10-6 and these two selective agents. For the most part, the mutants 3.6 x 1O-7 per cell per generation. That the mutants ap- cloned as well in the presence ofCPT-cAMP as in its absence. peared randomly in advance of selection was supported by Cloning efficiencies in Bt2cAMP were lower and more vari- both the presence of null populations and large variation in able both between experiments and among strains carrying numbers of mutants among the different populations (data the same mutation(s). In Bt2cAMP there was a trend favoring not shown). Table 2 summarizes sequence data for the 69 recovery of double over single mutant clones, but the differ- new spontaneous mutants isolated in these experiments. ences were small. These experiments suggested that selection Singe mutants containing the Trp-334 mutation were fre- with CPT-cAMP would result in both higher mutant recover- quent with both selective agents; they accounted for 75% of ies and less bias between single and double mutants. the CPT-cAMP-resistant isolates, suggesting the extraordi- The very high frequency of double mutants among spon- nary mutation frequency of 2 x 10-6 per cell per generation taneous Bt2cAMP-resistant mutants made us worry that the at a single C - G . Two Cys-332, Trp-334 double analog might have contributed to the mutagenic process. mutants were found among the CPT-cAMP-resistant clones, Several lines of evidence suggest that the Cys-332 mutation and one was found among the Bt2cAMP-resistant clones. confers little or no resistance to cAMP analogs: (i) protein Although double mutants were less frequent than in the kinase from double mutants has activation parameters indis- initial set of Bt2cAMP-resistant mutants characterized, they tinguishable from those of kinase from single Trp-334 mu- tants (10), (ii) the second mutation provides little if any cloning advantage over the Trp-334 mutation itself (Table 1), TABLE 2. Numbers of appearances of Trp-334 single and and (iii) kinase reconstituted from recombinant R subunit Cys-332, Trp-334 double mutations in independent spontaneous with the Cys-332 mutation has an activation constant only mutants resistant to cAMP analogsa slightly higher than that for kinase reconstituted with wild- type R subunit (see Fig. 3). Wild-type and Cys-332 mutants, Mutation then, would be expected to die shortly after exposure to Selective Expt Cys-332, Total agent Trp-334 Trp-334 Other' selective agents. A Trp-334 mutant, on the other hand, Single Double would survive treatment with Bt2cAMP and, if the analog were mutagenic, could acquire a second mutation during Bt2cAMP 1 1 1 1 3 clonal outgrowth. To test for this possibility, a Trp-334 2 8 0 6 14 mutant was subcloned in agarose under Bt2cAMP selection CPT-cAMP 1 9 1 7 17 conditions; 45 independent isolates were characterized by 2 30 1 4 35 R-subunit site either PCR and DNA sequence analysis of the a For the first experiment, freshly cloned populations of S49 subline U36 B region (12 isolates) or two-dimensional gel electrophoresis were grown to sizes of about 6 x 10- and recloned in the presence of either of radiolabeled R subunits (33 isolates). The subclones were CPT-cAMP or Bt2cAMP as described in Materials and Methods. From 30 all indistinguishable from the parental mutant; none had populations subjected to each selection, 17 yielded viable CPT-cAMP-resis- tant mutants and 3 yielded viable Bt2cAMP-resistant mutants. For the second acquired the Cys-332 mutation (data not shown). This exper- experiment, 42 freshly cloned populations of subline U36 were divided in two iment showed not only that Bt2cAMP does not promote after reaching a size of about 10' cells; the subpopulations were grown to sizes mutations at the Arg-332 codon but also that random occur- of about 106 and 2 x 106, respectively, for selection with CPT-cAMP or rence of the Cys-332 mutation is relatively infrequent (less Bt2cAMP. Single isolates from each population with viable mutant clones were analyzed for mutations in cAMP-binding site B as described in Materials than 0.025 per cell per generation). and Methods. Single (Trp-334) and double (Cys-332, Trp-334) mutants b Includes isolates with a variety of other mutations in R subunit as well as arise randomly and at high frequency. To determine the true those with no apparent R-subunit mutation (10). 770 STEINBERG AND GORMAN MOL. CELL. BIOL.

z a b 0 A C G T A C G T 1.0

O 0.8 w CD 0.6

...... * ...... ^ z 2 X 0.4 z 2 0.2 4: IL FIG. 2. Sequence analysis of subcloned amplified cDNA from a lo-,, 107c P Molo-, heterozygous double mutant. R-subunit site B DNA was amplified from mutant cDNA and subcloned into an M13 bacteriophage IcAMP], M vector; recombinant phage plaques were isolated and sequenced as described in Materials and Methods, using the internal positive- FIG. 3. cAMP-dependent activation of wild-type and mutant strand PST primer. Representative sequences shown are from an protein kinases reconstituted from recombinant R and C subunits. isolate with wild-type sequence (a) and an isolate with both Cys-332 Wild-type R subunits and R subunits with the mutations indicated and Trp-334 mutations (b). The wild-type sequence shown is were expressed in E. coli and reconstituted with purified recombi- TGCTGATGAATC*GTCCTC*GGGCTGCCACTGTGGT, with as- nant C subunit as described in Materials and Methods. Dilutions of terisks indicating mutated residues as before. Lanes are labeled as in the reconstituted material were assayed for protein kinase activity Fig. 1. without or with various concentrations of cAMP (and, for the Trp-334 mutant, with sufficient N6-benzoyl-cAMP to give full acti- vation). Data were normalized by subtracting the cAMP-indepen- dent activity and then dividing by the activity at saturating cyclic still accounted for more than 5% of all mutants that con- . tained 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 Kas for cAMP-dependent kinase activation by about 8- and analyzed from a number of independent populations and 65-fold, the Cys-332 and Gln-334 mutations had effects of only those from each population that were demonstrably only about 2-fold or less on kinase activation. different were retained and counted [16].) As could be predicted from the subcloning experiments of Table 1, DISCUSSION selection with CPT-cAMP in place of Bt2cAMP resulted in both a higher apparent mutation frequency and a slightly These results confirm a strong hotspot for spontaneous lower yield of double mutants. CG-*TA transition mutations at the first position of the The Cys-332 and Trp-334 mutations in double mutants are codon for Arg-334 of type I R subunit. This change was always physically linked. For double mutants from the set of found in almost 70% of 102 independent CPT-cAMP- or Bt2cAMP-resistant mutants isolated previously, the two- Bt2cAMP-resistant mutants analyzed in this and previous charge-unit acidic shifts in mutant R subunits provided studies (16, 22, 23). A second, closely linked CG--TA strong evidence for the presence of both mutations in the transition was found in seven of these mutants. The fre- same R subunit allele (16). Fo'r the three new double mu- quency of double mutants among heterozygous CPT-cAMP- tants, this linkage was confirmed by subcloning PCR prod- resistant isolates suggests a rate of occurrence of about 10-7 ucts into an M13 bacteriophage vector and sequencing per cell per generation. If one uses the very high rate of recombinant phage from a number of individual plaques. Trp-334 mutations as a standard for single mutations, the Representative (positive-strand) sequence patterns are expected rate for independent double mutations would be shown in Fig. 2. Each mutant gave recombinant phage that only about 4 x 10-12, or more than 4 orders of magnitude carried either the wild-type or the doubly mutated R subunit lower. Our failure to observe any spontaneous occurrences sequence; no phage carried only a single mutation. of a Cys-332 mutation alone in almost 200 S49 subclones Cys-332 and Gln-334 mutations have little effect on kinase analyzed- in these and other experiments (10, 16, 22, 23) activation. To assess the expected phenotypic effects of argues against the unlikely possibility that,mutations at the either a Cys-332 mutation alone or a second-position transi- Arg-332 codon occur randomly at a rate sufficient to account tion mutation in the Arg-334 codon (--*Gln), site-directed for their appearance by independent generation (almost 0.05 mutagenesis was used to introduce these mutations into an per cell per generation). The linked occurrence of the two R-subunit expre'ssion plasmid. The recombinant proteins mutations also is supported by the invariable colocalization were reconstituted with C subunit and assayed for cAMP- of the lesions in the same R-subunit allele and by the absence dependent kinase activity. Figure 3 shows the resulting of the Cys-332 mutation in subclones of a Trp-334 mutant activation curves and, for comparison, activation curves for that were isolated in the presence of Bt2cAMP. (Combining enzyme reconstituted with either wild-type R subunits or R the Bt2cAMP-selected mutants from these and previous subunits with a Leu-334 or Trp-334 mutation (both of which experiments [16], we found a total of 5 double mutants in 22 have given selectable phenotypes in S49 cells). In contrast to demonstrably independent sublines carrying the Trp-334 the Leu-334 and Trp-334 mutations, which shifted apparent mutation; because the earlier experiments discounted sub- VOL. 12, 1992 LINKED SPONTANEOUS CG--TA MUTATIONS AT CpG SITES 771 clones that might have been siblings, the total ofindependent dependent protein kinase, suggested the simpler explanation Trp-334-containing mutants in these experiments could be as that the second-position mutation did not confer a selectable high as 60. Depending on the estimate used for independent phenotype. A second-position transition mutation has been Trp-334-containing mutants, the finding of no double mu- found in the codon for Arg-332 (--His), but only in a tants in 45 Bt2cAMP-selected subclones of a Trp-334 mutant mutagen-induced double mutant (with the second mutation is significant to P values of about 0.006 to 0.077.) at residue 324); neither mutation at Arg-332 had a significant The Cys-332 and Trp-334 mutations both occur at CpG effect on kinase activation (Fig. 3) (4). If a precessive dinucleotides, which frequently are methylated in mamma- deaminase is responsible for the linkage of transition muta- lian DNA (7). From studies of the heat-induced deamination tions at CpG sites, the invariable association of Cys-332 of residues in DNA and of mutagenesis in wild-type rather than His-332 with the Trp-334 mutation suggests that and glycosylase-negative E. coli, it has been widely its precession is along a single DNA strand (either in assumed that deamination of 5-Me-C residues at CpG se- double-stranded DNA or in the single-stranded regions that quences would cause CG-*TA transitions and thereby con- arise during or replication). tribute to spontaneous mutation in mammalian systems (3, 5, Although multiple spontaneous mutations have been de- 8, 13, 15, 18). Extrapolation from published rates of 5-Me-C scribed in other systems, we have found only one other deamination in single- and double-stranded DNA at high reported instance of simultaneous transitions at closely temperatures and in single-stranded DNA at physiological linked CpG sites (20). That hotspots for double mutations temperature suggests that deamination of 5-Me-C residues in like those described here have not been observed in other double-stranded DNA at 37°C occurs at a rate of about 1.2 x somatic cell systems might be explained both by the rela- 107/h (or about 2 x 10-6/17-h S49 cell doubling time) (2). tively few systems that have been analyzed in depth and by Because of the probable attenuating effects of protection less fortuitous combinations of target sequences and selec- and/or repair mechanisms in vivo, this rate is almost surely tive conditions in those systems that have been studied. an overestimate of the rate at which such chemical events Furthermore, it is possible that the mutational pathway would cause mutations. The estimated rate of chemical leading to transition mutations at CpG sites is unusually deamination is thus barely sufficient to account for the active in S49 cells. observed rate of Trp-334 mutations; it is entirely incapable of explaining the high frequency of Cys-332, Trp-334 double ACKNOWLEDGMENTS mutations. The high frequency of linked mutations suggests either We thank Robert D. Cauthron and Marina M. Symcox for their that the two changes are linked mechanistically or that some substantial assistance in carrying out and preparing recombinant proteins for the reconstitution experiments of Fig. 3. We also thank process generates high local fluxes of mutagenic activity that Robert D. Ivarie and Lawrence A. Loeb for helpful suggestions and can simultaneously affect several sites within a restricted encouragement. target domain. Localty high concentrations of ionization This work was supported by grant DK37583 from the National products are unlikely to explain the mutations reported here, Institute of Diabetes and Digestive and Diseases. since the reactive species generated by irradiation induce transversions more frequently than transitions (11). Oxygen REFERENCES radicals produced in response to copper ions have been 1. Cambareri, E. B., B. C. Jensen, E. Schabtach, and E. U. Selker. shown to induce both single and double transition mutations 1989. Repeat-induced G-C to A-T mutations in Neurospora. in a bacterial system (25), but such a mechanism is also Science 244:1571-1575. inadequate to explain the results reported here; the copper- 2. Cooper, D. N., and M. Krawczak. 1989. Cytosine induced mutations had no apparent specificity for methyl- and the fate of CpG dinucleotides in genomes. Hum. ated or CpG sites, and the double mutations were all at Genet. 83:181-188. 3. Cooper, D. N., and H. Youssoufian. 1988. The CpG dinucleotide tandem bases. Error-prone DNA synthesis has been invoked and human genetic disease. Hum. Genet. 78:151-155. to explain multiple spontaneous mutations found both in 4. Correll, L. A., T. A. Woodford, J. D. Corbin, P. L. Mellon, and shuttle vector systems and in the phosphoribosyl- G. S. McKnight. 1989. Functional characterization of cAMP- transferase gene of a human carcinoma cell line (12, 21), but binding mutations in type I protein kinase. J. Biol. Chem. in contrast to those described here, the mutations attributed 264:16672-16678. to replication errors were more varied in type and spacing 5. Coulondre, C., J. H. Miller, P. J. Farabaugh, and W. Gilbert. and showed no preference for CpG sites. Because the 1978. Molecular basis of base substitution hotspots in Esche- Cys-332 and Trp-334 mutations are at CpG sites, we favor a richia coli. Nature (London) 274:775-780. mechanism that attributes them to deamination of 5-Me-C 6. De Jong, P. J., A. J. 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