Proc. Nail. Acad. Sci. USAVol. 82, pp. 2674-2678, May 1985Biochemistry

Growth-dependent expression of multiple species of DNAmethyltransferase in murine erythroleukemia cells

(DNA methylation/DNA replication/DNA repair/dye-ligand affinity chromatography)

TIMOTHY H. BESTOR AND VERNON M. INGRAMMassachusetts Institute of Technology, Department of Biology, Cambridge, MA 02139

Communicated by Ruth Sager, December 31, 1984

ABSTRACT Friend murine erythroleukemia cells werefound to contain three distinct species of DNA (cytosine-5-)-methyltransferase (DNA MeTase) whose relative proportionswere a characteristic function of the proliferative state of thecells. Rapidly proliferating cells contained a Mr 190,000 spe-cies of DNA MeTase (DNA MeTase III), whereas cells in thelate logarithmic/early plateau phase of cellular growth con-tained two species ofMr 150,000 and 175,000 (DNA MeTases Iand II); stationary phase cells contained primarily DNA Me-Tase I. The three species of DNA MeTase displayed structuralsimilarities, as determined by analysis of partial proteolysisproducts, and have similar de novo sequence specificities intransmethylation reactions involving purified enzyme and pro-karyotic DNA. The different relative proportions of the en-zymes in cells under different growth conditions suggest thatthe three species of DNA MeTase fulfill different roles in pro-cesses leading to the perpetuation of DNA methylation pat-terns.

The mammalian genome contains about 3 x 107 5' 5-methyl-deoxycytidyldeoxyguanosine (dm5CpdG) 3' dinucleotidesarrayed in tissue-specific patterns across 3 x 109 base pairsof DNA. These methylated sites can exert a strong influenceon the rate of transcription of adjacent sequences (reviewedin ref. 1). Changes in methylation patterns reflect changingpatterns of gene expression during development (2, 3) andare seen in the DNA of differentiating cultures of Friend mu-rine erythroleukemia (MEL) and F9 embryonal carcinomacells (4-7). Methylation patterns are established and main-tained through the action ofDNA (cytosine-5-)-methyltrans-ferase (DNA MeTase) (8); as is the case for the DNA repli-cating apparatus of higher cells, the molecular mechanism ofthe DNA methylating system is not well understood. Despitemuch effort, the means by which patterns of DNA methyl-ation are modulated during differentiation have not been re-vealed by in vitro studies of partially purified DNA MeTase,which typically displays little or no intrinsic sequence speci-ficity beyond the dCpdG dinucleotide (9, 10). Even thoughstimulated 30- to 100-fold by hemimethylated DNA sub-strates (9, 11), mammalian DNA MeTase displays inappro-priately high levels of de novo activity that would be expect-ed to degrade the precise patterns ofDNA methylation dur-ing the life-span of the organism, unless additionalconstraints operate within the cell; these constraints mustaffect enzyme specificity to prevent inappropriate methyl-ation of unmethylated dCpdG sites while permitting specificde novo methylation of certain sites, particularly during ear-ly embryogenesis (12). These constraints may be thought ofas unidentified nuclear factors that modify the action ofDNA MeTase through direct complex formation or by re-stricting access of the enzyme to specific regions of DNA.Additional evidence for the complexity of the process of

DNA methylation comes from the observations that methyl-ation of new DNA strands resulting from repair synthesis(13) seems to differ mechanistically from methylation of na-scent DNA strands resulting from DNA replication (14) andthat methylation of carcinogen-induced repair patches ismore rapid and complete in proliferating human fibroblaststhan in contact-inhibited cells (13). Terminally differentiatingF9 embryonal carcinoma (5-7) and MEL cells (4, 7), whichaccumulate large numbers of unmethylated and hemimethy-lated dCpdG sites, seem to experience a partial loss of theirability to methylate DNA during their last few cycles of divi-sion (7). These observations suggest that the DNA methylat-ing system is heterogeneous and may display different prop-erties according to the physiological state of the cell. Shiftingrelative proportions within groups of nuclear proteins of re-lated function such as the histones (15) and the DNA poly-merases (16) often accompany changes in growth state andare thought to represent adaptations to altered metabolic pri-orities. Previously, we have reported that MEL cells in thelate logarithmic/early plateau phase of cellular growth con-tain two species of DNA MeTase, which have been termedDNA MeTases I and 11 (9). We report here that MEL cellscontain a third distinct species of DNA MeTase, which ispresent in high levels only in rapidly growing cell popula-tions, and suggest roles for the three species of DNA Me-Tase in separate aspects of DNA metabolism.

MATERIALS AND METHODSPurification of DNA MeTase from MEL Cells. The PC4

subclone of line 745a was obtained from D. Housman of theMassachusetts Institute of Technology and grown to the in-dicated cell densities in 5 liters of medium contained in 10-liter spinner flasks as described (9). DNA MeTase was ex-tracted from Triton X-100-washed nuclei with 0.25 M NaCland partially purified by chromatography on DEAE-Sepha-cel (9). Active fractions were pooled and brought to 30% sat-uration in (NH4)2SO4; the precipitate was collected by cen-trifugation and discarded. The supernatant was made 60%saturated in (NH4)2SO4 and the precipitate (which containedall of the DNA MeTase activity) was redissolved in "M buff-er" (0.02 M Tris HCl, pH 7.4/2.7 M glycerol/0.5 mM dithio-threitol/5 mM Na3EDTA/0.2 mM phenylmethylsulfonyl flu-oride) and immediately chromatographed on Cibacron blue-agarose as described (9), except that the gradient was 0-2 MNaCl in M buffer. When further purification was required,peak fractions from the Cibacron blue-agarose column werediluted with an equal volume of 2.5 M (NH4)2SO4 and ap-plied to a 1 x 10 cm column of phenylagarose (Sigma) equili-brated with 1.25 M (NH4)2SO4 in M buffer containing only1.35 M glycerol. Bound proteins were eluted with a 1.25-0 Mgradient of (NH4)2SO4. Essentially homogeneous prepara-tions could be obtained by chromatography of the resulting

Abbreviations: DNA MeTase, DNA (cytosine-5-)-methyltrans-ferase; MEL, murine erythroleukemia; dCpdG, deoxycytidyldeoxy-guanosine.

2674

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Proc. NatL. Acad. Sci. USA 82 (1985) 2675

peak fractions on hydroxyapatite as described (9). Becauseof the severalfold increase in total DNA MeTase activity thatfollows DEAE-Sephacel chromatography (presumably asthe result of the removal of an inhibitory substance), mean-ingful estimates of the total purification factor could not beobtained. Enzyme activity was assayed by adding 5 Al ofsolution to be assayed to 200 A.l of M buffer containing 0.1AM S-adenosyl-L-[methyl-3H]methionine (12 Ci/mmol; 1 Ci= 37 GBq; Amersham) and 10 Ag of poly(dG-dC) (P-L Bio-chemicals) per ml. Trichloroacetic acid-insoluble radioactiv-ity was determined as described (9). One unit of enzyme ac-tivity incorporated 1 pmol of 3H in 1 hr at 370C and, at thecounting efficiency used, was equivalent to 8.9 x 103 cpm.Protein composition of chromatographic fractions was deter-mined by electrophoresis of aliquots on NaDodSO4/6%polyacrylamide gels. Samples were prepared by dilutionwith 1 vol of 100 ug of sodium deoxycholate per ml and pre-cipitation with trichloroacetic acid at a final concentration of10%. Precipitates were collected by centrifugation at 12,000x g for 15 min, washed once with acetone, and redissolvedin NaDodSO4 loading buffer (9). To correct for differing cellnumbers among cultures grown to different densities, thevolume of buffer used for elution of DEAE columns wasadjusted to give a constant value for the ratio of applied pro-tein versus total eluate volume.

Peptide Mapping of Partial Proteolysis Products. Separat-

I-

I-

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ed, purified DNA MeTase species were subjected to finalpurification on NaDodSO4/6% polyacrylamide gels. Peptidemaps were obtained by partial proteolysis during coelectro-phoresis of protein in excised gel slices with 25 ng of Staph-ylococcus aureus V8 protease on NaDodSO4/12% poly-acrylamide gels (17).

Determination of the de Novo Sequence Specificities ofMELCell DNA MeTase. Plasmid pLP32 (18) was enzymaticallymethylated to a specific activity of about 8 x 104 cpm/,ug byincubation with S-adenosyl-L-[methyl-3H]methionine andDNA MeTase. Plasmid DNA was cleaved with Hae III togive fragments with a median dCpdG content of 16 (19), thefragments were separated by electrophoresis through an 8%polyacrylamide gel, and a fluorogram was prepared afterequilibration of the gel with EN3HANCE.

RESULTS

Multiple Species of DNA MeTase in MEL Cells. Cibacronblue-agarose has been shown to provide a sensitive meansfor the chromatographic separation of two species of DNAMeTase from salt extracts of detergent-purified MEL cellnuclei (9); in that study, cells were grown to near-saturatingdensities to maximize cell yields (MEL cells grow with ageneration time of 12 hr to reach a final density of 1-1.5 x106 per ml in 10-liter spinner flasks). In the present study,

A10-

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10-

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FIG. 1. Growth state determines types ofDNA MeTase in MEL cells. (A) Cells harvested in mid-logarithmic phase (4 x 105 cells per ml). (B)Cells harvested upon reaching 106 cells per ml (late logarithmic/early plateau phase). (C) As in B, except that incubation was continued for 24 hrafter reaching saturating density of 106 cells per ml. (D) As in A, except that cycloheximide was added to a final concentration of 35 ,ug/ml at 10hr prior to harvest. The concentration of morphologically normal cells declined from 2.0 x 106 to 1.3 x 106 cells per ml during antibiotictreatment. (E) Dimethyl sulfoxide-induced cells. Cells at an initial density of 2 x 104 cells per ml were treated for 96 hr with 250 mM dimethylsulfoxide, by which time 75% of the cells were positive for hemoglobin, as assessed by benzidine staining. In A-E, arrowheads indicate bandscorresponding to DNA MeTase I (Mr 150,000), which elutes from Cibacron blue-agarose first, followed by DNA MeTases II (Mr 175,000) andIII (Mr 190,000). The amounts of protein applied to the gels were adjusted to allow optimal visualization of DNA MeTase; apparent bandintensities and amounts of enzyme activity in Fig. 1 are not directly comparable. Variability in background proteins apparent in the Coomassieblue-stained gels is an artifact of the isolation procedures.

Biochemistry: Bestor and Ingram

2676 Biochemistry: Bestor and Ingram

DNA MeTase activity purified from cells harvested in themidlogarithmic phase of growth (0.5-5 x 105 per ml) wasfound to reside in a polypeptide ofMr 190,000 (DNA MeTaseIII), which could be separated from DNA MeTases I and IIby chromatography on Cibacron blue-agarose (Fig. 1 A andB). When cells were grown to saturating density and incubat-ed for a further 24 hr, the bulk of the activity was found to bein the form of DNA MeTase I, enzyme III being undetect-able and enzyme II present in reduced amount (Fig. 1C). Noenzyme activity or protein bands at the expected positionswere observed when extracts of dimethyl sulfoxide-inducedMEL cells were analyzed by chromatography on Cibacronblue-agarose (Fig. 1E), although low levels ofDNA MeTaseactivity were detectable through DEAE-cellulose chroma-tography.

Total DNA MeTase activity per 106 cells did not differ bymore than 2-fold among logarithmic phase, late logarith-mic/early plateau, and stationary phase cell populations; ac-tivity recovered from cycloheximide-treated cells was lowerby a factor of about 4 (data not shown).The Three ]DNA MeTases Have Similar Structures and in

Vitro Enzymatic Properties. Each enzyme was further puri-fied by hydrophobic interaction chromatography on phenyl-agarose as described in Materials and Methods; essentiallyhomogeneous preparations of each enzyme could be ob-tained by chromatography ofpeak fractions from the phenyl-agarose column on hydroxyapatite as described (9) (see Fig.4). Peptide maps were obtained by partial proteolysis inpolyacrylamide gels as described by Cleveland et al. (17).Fig. 2 shows that the three enzymes yielded very similarcleavage products, indicating substantial homology.Mammalian DNA methyltransferases, including DNA Me-

Tases I and II, have been demonstrated previously to trans-fer methyl groups from S-adenosyl-L-methionine to DNA atan initial rate defined by the dCpdG content of the DNAsubstrate (9, 10). A sensitive method for evaluating the denovo sequence specificity of a DNA MeTase consists ofmethylation of aDNA of defined sequence in the presence ofS-adenosyl-L-[methyl-3H]methionine, cleavage of the DNAwith restriction endonuclease to produce small DNA frag-ments, and electrophoretic separation of the radioactive

l II 111

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FIG. 2. One-dimensional peptide maps reveal substantial homol-ogy among the three forms of DNA MeTase. Numerals along topindicate type ofDNA MeTase (I, II, or III), and arrowheads indicateprominent Coomassie blue-stained bands that are in common amongall three forms ofDNA MeTase. Although certain differences in thepatterns are also apparent, these do not necessarily reflect differ-ences in primary sequence because of the very large number of par-tial proteolysis products that can be generated from large proteins.Numerals on the left indicate Mr X 103.

I 11 III

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FIG. 3. Sequence specificity of MEL cell DNA MeTases. Plas-mid pLP32 was enzymatically methylated with the DNA MeTasespecies indicated at the top. DNA was cleaved with Hae III andfragments were separated on an 8% polyacrylamide gel. Relativeband intensities can be seen to be very similar across the fluoro-graphic image of the gel, indicating that DNA MeTases I, II, and IIIhave very similar sequence specificities when performing de novomethylation. Lengths of DNA fragments in base pairs are given onthe left.

DNA fragments in agarose or polyacrylamide gels. Differ-ences in sequence specificity among different enzymes willbe observed as differences in specific radioactivities ofDNAfragments of the same sequence (9).A fluorogram displaying the results of such an analysis is

shown in Fig. 3, in which the substrate DNA was plasmidpLP32 [derived by insertion of (dG-dC)16 into the BamHIsite of pBR322] (18). Relative band intensities are very simi-lar across the fluorographic image of the gel, indicating thatthe de novo sequence specificities of the three enzymes arevery similar. The band at 104 base pairs contains the (dG-dCh6 insert and is especially heavily labeled, reflecting thehigh dCpdG density of this fragment. The preference ofDNA MeTase III for hemimethylated dCpdG sites was alsovery similar to that of enzymes I and II (about 30-fold greateractivity than on unmethylated substrates), as determined byusing the synthetic copolymer substrate poly(dG-dC,dm5C)containing 80 mol% m C (data not shown) (9).

Relationship of the Three Species ofDNA MeTase. The sim-ilar physical and enzymatic properties of the three forms ofMEL cell DNA MeTase suggested that they may be derivedby post-translational processing of a common precursor;proteolytic processing of the 190,000 Mr form to the 175,000and 150,000 Mr forms is the most obvious route. Growth ar-rest (as a result of growth to saturating density or othercause) might provide the stimulus for such a conversion. Totest this hypothesis, MEL cells in midlogarithmic phase(which contain primarily DNA MeTase III) were treatedwith 35 pg of cycloheximide per ml to arrest cell growth andhalt protein synthesis. After 10 hr of treatment, cells wereharvested and DNA MeTase was purified. If arrest of cellgrowth were to trigger conversion of DNA MeTase III to Iand II by activation of a pre-existing protease, an accumula-tion of enzymes I and II should have been observed; instead,DNA MeTase III remained the predominant form (Fig. ID).Synthesis of new protein seems to be required for the con-version of DNA MeTase types, although it cannot be con-cluded from these data if it is de novo synthesis of DNAMeTase itself or a factor involved in post-translational pro-cessing that is required. However, since substantial celldamage and lysis were incurred by cycloheximide-treated

Proc. Nad Acad Sci. USA 82 (1985)

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Hi

Proc. NatL. Acad. Sci. USA 82 (1985) 2677

cells (as reflected by a decrease of 35% in the number ofmorphologically normal cells during treatment), it can beconcluded that the changes in relative proportion of DNAMeTase types is not an artifactual event occurring as a resultof cell death or lysis; this conclusion is supported by the ob-servation that cells grown to late logarithmic/early plateauphase contain very little DNA MeTase III (Fig. 1B) but arefully viable after dilution into fresh medium (data notshown).There is some evidence that proteolytic processing is in-

volved in the conversion of DNA MeTase II to MeTase I,since incubation of enzyme II at 40C resulted in the appear-ance of a small amount of a polypeptide that comigrated withDNA MeTase I during NaDodSO4/polyacrylamide gel elec-trophoresis (Fig. 4B), presumably as a result of cleavage by aprotease present as a trace contaminant. DNA MeTase IIappears to undergo additional modifications in vivo, sincesome purified preparations form closely spaced doublet ortriplet bands on NaDodSO4/polyacrylamide gels (Fig. 4B);the reason for this is not known. Since DNA MeTase II ispresent only transiently in cells undergoing cessation ofgrowth and because some conversion of DNA MeTase II toDNA MeTase I was observed in vitro (Fig. 4B), DNA Me-Tase I may represent the biologically relevant form in noncy-cling cells. Evidence for heterogeneity of DNA MeTases Iand III has not been observed, and proteolysis to lower mo-lecular weight forms did not occur even after storage of par-tially purified preparations of these enzymes at 40C for 30days (data not shown).

Proteolytic degradation of DNA MeTase III during en-zyme purification could explain the observed multiplicity ofDNA MeTase species. This explanation can be excluded,since the relative proportions of the three enzymes were re-producible functions of the growth phase of the cells and ofno other variable; variations in the time elapsing betweenpreparation of crude nuclear extracts and DEAE-Sephacelchromatography (the purification step during which proteol-ysis might be expected to be most likely) of from 2 to 6 hr did

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FIG. 4. Partial conversion of purified DNA MeTase II to a poly-peptide that comigrates with DNA MeTase I. (A) DNA MeTase IIanalyzed by NaDodSO4/polyacrylamide gel electrophoresis imme-diately after isolation. (B) Lane 1 is purified DNA MeTase I, lane 2contains protein size standards (myosin, f-galactosidase, and phos-phorylase b; numerals indicate respective Mr X 10-3), and lane 3contains DNA MeTase II (from the same preparation as the materialin A) after incubation for 48 hr at 4TC. A new band has appeared,which comigrates with DNA MeTase I (lower arrow) and which ispresumably the result of proteolysis. Purified DNA MeTase II oftensplits into doublet or triplet bands, as shown here. (C) Lane 1 con-tains purified DNA MeTase III, and lane 2 contains protein sizestandards. Arrow indicates 190,000 Mr DNA MeTase III. Each lanereceived 2 ,ug of protein.

not alter the relative amounts of the three enzymes, andomission of the serine protease inhibitor phenylmethylsul-fonyl fluoride did not alter enzyme ratios. In addition, thecatalytically active proteolytic fragment ofDNA MeTase re-sulting from trypsin treatment of enzyme prepared fromKrebs II ascites cells has a sedimentation coefficient thatindicates a molecular weight substantially lower than that ofthe enzymes described here (20). We conclude that thegrowth-phase dependence of the relative amounts of DNAMeTases I, II, and III reflects an in vivo phenomenon. Be-cause of the labor and expense required to obtain the nmolamounts of purified protein required for studies of proteinsequence and structure, the use of specific antibodies inpulse-chase experiments and nucleic acid hybridizationprobes to identify DNA MeTase mRNA species will proba-bly be necessary to determine the precise relationship of thethree species of DNA MeTase described here.

DISCUSSIONIt has been demonstrated here that MEL cells contain threedistinct species of DNA MeTase whose relative proportionsare defined by the proliferative state of the cells. This find-ing, together with data from studies of DNA replication andDNA methylation in vivo, immediately suggests that the dif-ferent forms ofDNA MeTase participate in different aspectsof DNA metabolism.DNA polymerase a, the form ofDNA polymerase thought

to be primarily responsible for DNA replication synthesis,shows behavior strikingly similar to that reported here forDNA MeTase III in that each enzyme is present in apprecia-ble amounts only in rapidly proliferating cell populations(16). This suggests that DNA MeTase III is likely to be re-sponsible for preserving methylation patterns by acting athemimethylated sites in newly replicated DNA and is theform of DNA MeTase that is likely to be most analogous infunction to the hypothesized "maintenance" DNA methyl-transferase (21). Following this line of reasoning, we suggestthat DNA MeTases I and II, which are the predominant spe-cies in noncycling cells, may be involved in the methylationof DNA repair patches. The predominant form of DNA syn-thesis in noncycling cells is repair synthesis, which differs inimportant respects from replication synthesis (13), althoughDNA methylation in noncycling cells should also be of themaintenance type, since hemimethylated sites should also bepresent in regions of DNA that have undergone repair. Arole in DNA repair is attributed to DNA polymerase / on thegrounds that it is the major nuclear DNA polymerase in non-cycling cells (22), where DNA MeTases I and II are the ma-jor DNA methylating enzymes. However, the convenient as-signment of replication synthesis to DNA polymerase a andrepair synthesis to DNA polymerase ,B is probably unjustifi-able, since little direct evidence for these roles is availableand because repair of high-dose UV damage in human fibro-blasts is sensitive to aphidicolin [a drug which is thought tobe a specific inhibitor of DNA polymerase a and y (23)],suggesting involvement of DNA polymerase a or relatedproteins in repair synthesis (24). Although the roles of differ-ent DNA polymerases cannot be stated with certainty (25),the growth-dependent changes in levels of the three speciesof DNA MeTase are best accounted for by postulating dis-tinct roles in the processes of post-replication and post-re-pair DNA methylation; the disparate efficiencies of theseprocesses are also explicable in this way.The above proposal requires that at least two separate

pools of DNA MeTase exist in the nucleus-one pool in-volved in the methylation of newly replicated DNA and lo-cated near the replication fork (resulting in a higher effectiveconcentration of DNA MeTase or directly coupled replica-tion and methylation) and the other responsible for methylat-

Biochemistry: Bestor and Ingram

2678 Biochemistry: Bestor and Ingram

ing repair patches and presumably distributed throughoutthe nucleus. The relative sizes of these pools of DNA Me-Tase would be expected to reflect the predominant form ofDNA synthesis (replication or repair) occurring in the cell.The differing effective concentrations of DNA MeTase ofdifferent pools could explain why post-replication methyl-ation is essentially complete within minutes (14), while un-methylated sites in repair patches can persist for many hours(13). This hypothesis is obviously consistent with the experi-mental results described in this paper, since density-depen-dent changes in the predominant form of DNA synthesis(replication synthesis in growing cells and repair synthesis innongrowing cells) are accompanied by an altered spectrumofDNA MeTase species. A testable hypothesis arising fromthis line of thought is that the DNA MeTase species reportedto be associated with DNA replication complexes (26, 27) isDNA MeTase III (see below). However, the situation maybe complicated somewhat by the observation that methyl-ation of repair patches after carcinogen treatment is less effi-cient in contact-inhibited than in rapidly growing human fi-broblasts in which replication synthesis had been inhibitedby treatment with hydroxyurea (13).

It is interesting that a DNA MeTase activity has been re-ported to be present as part of a large particle containingenzymes involved in DNA replication, including DNA poly-merase a, DNA topoisomerase, and enzymes involved indeoxynucleotide biosynthesis (26). In another study, ion-ex-change chromatography was used to resolve four soluble en-zyme complexes of DNA polymerase a and other DNA rep-lication factors (27); two of the forms contained DNA Me-Tase, although the level of DNA MeTase activity was lowand the proportion of total DNA MeTase activity present inthe form of complexes was not reported. Arguing againsttight linkage of DNA polymerase and DNA MeTase is thefact that different experimental approaches uniformly showthat some DNA methylation occurs some distance behindthe replication fork (14, 28); in transfected eggs of Xenopuslaevis, hemimethylated DNA can be methylated in the ab-sence of DNA replication (29). These observations are diffi-cult to reconcile with the reported physical association ofDNA polymerase and DNA MeTase, unless the existence ofmore than one functional pool of DNA MeTase is postulat-ed.The influence of induced differentiation on the DNA

methylating apparatus of MEL cells is not completely clear,as cells induced to undergo differentiation by treatment withdimethyl sulfoxide contained only a small amount of DNAMeTase activity (following DEAE-Sephacel chromatogra-phy, less than one-third of that of an equal number of station-ary-phase cells), which could not be recovered from columnsof Cibacron blue-agarose (Fig. 1E), preventing identificationof the DNA MeTase species present in induced cells. Thebasis for the instability of partially purified DNA MeTasefrom differentiated cells is not understood but could be aresult of the increased levels of degradative enzymes in dif-ferentiating erythroid cells (30). The partial loss of recover-able DNA MeTase activity correlates with the demethyl-ation of the genome observed in differentiating MEL (4, 7)and F9 embryonal carcinoma cells (5-7), in which the num-ber of hemimethylated and demethylated sites exceeds bymany thousandfold the number of genes whose expression isknown to change during in vitro differentiation.

Errors in the inheritance of methylation patterns may haveserious consequences for cellular differentiation and gene

expression (31); an understanding of the mechanism ofDNAmethylation is important for these and other reasons. Defini-tion of the number and properties of cellular DNA MeTaseand the determination of the relationship ofthese enzymes toother DNA-metabolizing enzymes would represent a basisfor understanding the mechanism by which methylation pat-terns are established, maintained, and modulated during thelife of the organism.

This research was supported by Public Health Service GrantAM13945 from the National Institutes of Health. T.H.B. is a Fellowof the Leukemia Society of America.

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