Mutation Research, 150 (1985) 77-84 77 Elsevier

MTR 02019

Methylation-induced blocks to in vitro DNA replication

Karen Larson, Janet Sahm, Robert Shenkar and Bernard Strauss Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637 (U.S.A.)

(Received 3 January 1985) (Accepted 7 January 1985)

Summary

Single-stranded primed M13mp2 templates and double-stranded templates were treated with either dimethyl sulfate (DMS) or N-methyl-N'-nitro-N-nitrosoguanidine and used for DNA synthesis in vitro. Methylation inhibits the ability of the molecules to serve as templates. When either E. coil DNA polymerase I or AMV reverse transcriptase were used as polymerases, DNA synthesis terminated one nucleotide 3' to the site of adenine residues in the template. Heating of the templates resulted in the appearance of additional termination bands one nucleotide before the site of G's in the template. We assume that methylated A's but not methylated G's are blocks to in vitro DNA synthesis and that heating converts a portion of the sites of methylated G to AP sites which are blocks to synthesis.

Termination of DNA synthesis as the result of the formation of large mutagenic adducts or of the production of AP (apurinic/apyrimidinic) sites in DNA occurs one nucleotide before (3' to) the site of template damage (Moore and Strauss, 1979; Strauss et al., 1982; Sagher and Strauss, 1983). The adducts studied have either produced a drastic change in nucleotide structure as in the formation of N-guanin-8-yl-acetyl-2-aminofluorene (Rabkin and Strauss, 1984) or have resulted in a non-in- structive AP site by removal of a pyrimidine (Sagher and Strauss, 1983). Since relatively small adducts, such as methyl groups, play a large role in the phenomena of mutagenesis (see Walker, 1984) we thought it important to determine the ability of such groups to serve as chain termina-

Send communications to: Dr. Bernard Strauss, Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 E. 58th Street, Chicago, IL 60637 (U.S.A.).

Abbreviations: DMS, dimethyl sulfate; MNNG, N-methyl- N '-nit ro-N-nitrosoguanidine.

tors. A method of approaching the problem is to methylate DNA with a simple alkylating agent and determine its ability to serve as a template for in vitro DNA synthesis. The obvious difficulty is that methylating agents produce a spectrum of 10-13 different changes in nucleotide structure (Singer and Grunberger, 1983). Traditional ap- proaches do not indicate which are the critical lesions.

The methodology we have described for study of the termination of reactions (Moore and Strauss, 1979; Rabkin and Strauss, 1984) has utilized re- agents which yield a simple pattern of reaction. A DNA template of defined nucleotide sequence is reacted with mutagen and then primed for DNA synthesis. Termination of synthesis occurs exactly one nucleotide before the altered template nucleo- tide. The availability of this collection of data with materials producing simple reaction patterns prompted us to attempt this study. We wanted to know whether understandable termination pat- terns could be obtained with alkylating agents as varied in their reaction products as dimethyl sulfate

0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

78

(DMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We were also interested in determining whether the pattern of chain termination seen with primed single-stranded DNA templates was the same as that seen when double-stranded templates, which may mimic the natural situation more closely, were used. In this investigation we show that the termination sites after treatment with either dimethyl sulfate or M N N G occur before A's on the template, whether single- or double- stranded. This in vitro result may have some bio- logical significance.

Materials and Methods

DNA. Bacteriophage M13mp2 viral strand and replicative form I DNA were prepared as de- scribed by Sagher and Strauss (1983).

Preparation of primed single strand templates. A short primer (123bps) was prepared by cleaving M13mp2 RFI DNA successively with EcoRI (New England Biolabs) and Pvu I (Boehringer Man- nheim) under conditions recommended by the supplier. The two resulting fragments were sep- arated by electrophoresis through a 5% poly- acrylamide gel. After ethidium bromide staining, bands containing DNA were cut from the gel. DNA was electroeluted into t r is -HC1/EDTA, pH 7.6 in dialysis bags. After recovery from the bag, DNA was butanol-extracted, precipitated with sodium acetate/ethanol and suspended in 10 mM Hepes/1 mm EDTA, pH 7.5. The priming frag- ment was denatured at 100°C for 5 min in the presence of 100 mM KC1 and quenched in ice. Viral strand DNA was added to a concentration of 50/~g/ml. Hybridization was for 90 rain at 65°C. The ratio of double stranded to single-stranded DNA (mole/mole) was 1.2 to 1.

Preparation of uniquely nicked, double-strand template. The priming strand was created by cleaving replicative form I M13mp2 DNA with EcoR1 (New England BioLabs), an enzyme which cleaves the molecule only once. Cleavage was car- ried out under the conditions recommended by the supplier. The DNA was chloroform-phenol ex- tracted, precipitated with sodium acetate, ethanol and resuspended in 10 mM Hepes/1 mM EDTA, pH 8.0. The linearized molecules (RF III) were then denatured at 100°C for 5 min and the mix-

ture quenched in ice. Viral strand DNA was added and the salt concentration adjusted to 100 mM with KC1. Hybridization was for 30 min at 65°C with subsequent slow cooling. The molar ratio of RF III to viral strand was 0.5:1. Hybridizations were carried out at a viral strand concentration of 75/x g/ml.

Template DNA was purified from contaminat- ing single strands and from DNA which had rean- nealed to form RF III (Fig. 1). The hybridization mixture was loaded onto a 1% agarose slab gel and electrophoresed at 80 V for 5 h. One side of the gel was ethidium bromide stained and the band con- taining nicked circular molecules was marked. A corresponding band from the main gel was cut out and placed into a dialysis bag with a small amount of t r i s -HC1/ace ta te /EDTA, pH 7.6. The DNA was electroeluted from the agarose at 100 V for 1.5 h, removed from the bag and concentrated by butanol extraction. The sample was extracted once with phenol, twice with chloroform/phenol and twice with ether, then precipitated twice with

I-- >- ~ o-) -r- I~.

n.d.s.

n .d . s . : n i c k e d a o u b l e s t r a n d circles

Fig. 1. Purification of uniquely nicked double-stranded tem- plate. Standards in lane one include (in descending order) M13mp2 RF III, RF I and viral strand. In lane two the products of the hybridization of viral strand DNA with EcoR1 linearized M13mp2-RF I. The top band is the desired hybrid. Lane 3: uniquely nicked template after purification. Hybridiza- tion conditions are given in Materials and Methods.

sodium acetate/ethanol. The final pellet was re- suspended in 10 mM Hepes /1 mM EDTA, pH 8.0.

Dimethy lsu l fa te treatment . Primed single- strand templates were treated with 5 mM dimethyl sulfate (DMS) in methanol for 30 min at 25°C. The DNA concentration was 25/~g/ml. Reactions were carried out in 200 mM sodium cacodylate buffer, pH 7.0, and were stopped by the addition of fl-mercaptoethanol to 200 mM. Treated D N A was precipitated twice by the addition of sodium acetate/ethanol. Double-stranded templates were reacted similarly, but with 10 mM DMS at a D N A concentration of 50/~g/ml . In order to quantitate the number of lesions produced in D N A under these conditions, reactions were performed with [3H]dimethyl sulfate (New England Nuclear). Single-strand template had 65 adducts/molecule; double-stranded template had 150 adducts /mole- cule.

D N A polymerase reactions. D N A polymerase reactions contained 0.24/~g primed single-stranded DNA, 0.2 units E. coli D N A polymerase Klenow fragment (Boehringer Mannheim) or 8 units AMV reverse transcriptase (Life Sciences), 0.6 #M[et- 32 P]dATP(400 C i /mM) , 50 #M each dGTP, dCTP, dTTP, 50 mM tris-HC1, pH 8.0, 5 mM dithio- threitol, 8 mM MgC12 in 6 /tl. Reactions were incubated 15 min at 30°C for pol I Kf or at 35°C for AMV reverse transcriptase. At the conclusion of the initial reaction period, dATP was added to 80 /~M and incubation was continued for an ad- ditional 15 min.

For reactions utilizing double-strand templates, 0.1 /~g D N A was incubated with 8 units AMV reverse transcriptase, 0.6 /tm each [a-32p]dATP, [a-32p]dGTP, [a-32p]dCTP, [a-32p]dTTP (approx.

400 C i / m m o l e each), 50 mM tris-HC1, pH 8.0, 100 mM KC1, 5 mM dithiothreitol, 8 mM MgC12 for 10 min at 35°C. All four dNTP's were then added to 100 /~M each and incubation was con- tinued for an additional 20 min except as indi- cated below. The polymerase was inactivated at 65°C, D N A was restricted with EcoR1 for 45 min at 37°C and then electrophoresed on 14% denatur- ing polyacrylamide gels as described previously (Sagher and Strauss, 1983). Gels were autoradio- graphed using Dupont Cronex film.

The enzymes were assayed for nuclease activity

79

by !ncubating the product of first-stage reactions on an N-acetoxy-N-acetyl-2-aminofluorene-treated template (Rabkin and Strauss, 1984) with enzyme and either dCTP or water. As expected for an enzyme with an active 3' > 5' nuclease, incubation with pol I (Kf) in the absence of dNTP's led to extensive degradation. In contrast, incubation with AMV reverse transcriptase in the absence of dNTP's resulted in no change in the substrate at some termination sites and only minor change at others (data not shown).

Results

DMS-treated D N A is less efficient as a tem- plate than is untreated, primed D N A with either E. coli pol I ( K f ) o r with AMV reverse tran- scriptase (Fig. 2). AMV reverse transcriptase is less

F)

X

0 0

0 E

Z

P011 (Kf) (s.sJ

- AMVr.t . (d.s.)

A M V r.t.(s.~)

o 12 o CO 20 40 60 ( 20 40 60

T I M E I N C U B A T E D ( r n i n )

Fig. 2. Time course of nucleotide incorporation on control and DMS-treated templates. Double-stranded (d.s.) templates with E. coli pol I (holoenzyme) or with AMV reverse transcriptase. Single stranded (s.s.) templates with E.. coli Pol I (Kf) or AMV reverse transcriptase. Open circles non-treated (control) tem- plates. Closed circles: DMS-treated. d.s., treated with 10 mM DMS; s.s. with 5 mM DMS both for 30 min at 25°C, pH 7.0. Reaction mixtures are descibed in Materials and Methods. All reactions utilized [32p]dATP as the labeled nucleotide. The specific activity for [ 32 P]dATP in the reaction mixtures varied slightly from day to day and averaged 6.2 x 106 cpm//tM for the s.s. and 1.1 × 10 7 cpm/ttM for the d.s. reaction mixtures.

80

efficient in elongating a primer on a single-stranded template than is pol I (Kf). AMV reverse tran- scriptase is unable to catalyze appreciable synthe- sis on a nicked double-stranded template as would be expected from its lack of a 5' > 3' exonuclease and from its very limited ability to strand displace (Leis and Hurwitz, 1972). In spite of the limited reaction, we have been able to obtain clear termination patterns on alkylated double-stranded templates, since the enzyme is able to synthesize up to 200 nucleotides under our conditions. The average distances for synthesis on single- and dou- ble-stranded alkylated templates (Fig. 2) are re- lated to differences in the average number of alkyl groups added. Assuming that only alkyl groups on the template strand are effective in blocking

AMY rev.tran~

. . . . Po! I (~ ) Fig. 3. Termination of synthesis on DMS treated single-stranded templates using either AMV reverse transcriptase or E. coli polymerase I (Klenow fragment), dd lanes indicate the dideoxy chain terminators ddATP, ddGTP, ddTTP and ddCTP. Num- bering is from the EcoR1 site. Reaction mixtures using DMS-treated, methanol-treated or non-treated templates are shown. Ht, heated at 65°C for 1 h. ( - ) non-heated template.

synthesis, the ratio of nucleotides added to single- and double-stranded templates is in direct propor- tion to the relative number of adducts. We have independent evidence that lesions on the non-tem- plate strand do not terminate synthesis (Larson, in preparation). We should be able to calculate the number of chain-blocking lesions from the average number of nucleotides added per DNA molecule. Such a calculation assumes that all DNA circles have been primed, accurately reflecting the, case for purified double-stranded templates. However, our previous results suggest this is a poor assump-

81

tion for primed single stranded templates (Moore et al., 1980).

Treatment of single-stranded DNA with DMS results in a substrate which blocks DNA synthesis at multiple positions when either E. coli pol I (Kf) or AMV reverse transcriptase is used as the poly- merase (Fig. 3a,b). We used treated DNA either directly or after heat induced depurination (65°C for 1 h). The pattern of bands is simplest with the unheated samples. With either AMV reverse tran- scriptase (Fig. 3a) or pol I (Kf) (Fig. 3b) stops occur one nucleotide before A's or C's in the template. With AMV reverse transcriptase, we ob- serve stops before A's at positions 21, 22, 25, 27, 29, 34, 35, 37, 38, 40, 45, 51 and 52, and before C's at position 43. With pol I (Kf) (Fig. 3b) stops before A's occur at positions 21, 22, 25, 27, 29, 34, 35, 37, 38, 40, 51, 52 and before C's at positions 28 and 36. A stop before G appears at position 46 and a weak stop occurs at position 39 before a T.

The methylated and heated templates result in a more complex pattern of termination with ad-

ditional bands appearing in the AMV reverse tran- scriptase reaction at positions 23, 24, 39, 42, 46, 47, 48, 49 and higher on the template. These and additional bands shown in Fig. 3 occur before G's in the template in 12 cases, before T's in 6 cases

a n d in one case before a C. 12 out of 18 of the additional stops occur opposite a purine. A similar pattern of termination after heating is seen with pol I (Kf) (Fig. 3b). Additional stops are seen, for example at positions 23, 24, 42, 43, 44 (light), 45, 47, 48, 49. In 10 cases these new bands occur before G's; 11 of 16 total stops occur opposite purines.

The role of A as a termination site is seen even more clearly in double-stranded alkylated tem- plates (Fig. 4). Stops are seen at positions 20, 21, 22, 25, 27, 29, 34, 35, 37, 38, 40, 45 and 46. All of these stops occur one nucleotide preceding an A in the templat~ strand. In contrast with the results on single-stranded template, and as expected consid- ering the lack of reactivity of cytosine in double- stranded DNA with alkylating agents, there are no

Fig. 4. Termination of synthesis on DMS-treated double-stranded templates using E. coli polymerase I. dd lanes as in legend to Fig. 3. Synthesis was for 10 min at room temperature with all four [32p]dNTP's at 0.6/~M followed by synthesis for an additional 5, 20 or 80 min in the presence of 100 # M unlabeled dNTP's. Numbering is from the EcoR1 site.

82

termination bands before C (position 43, Fig. 3). Similar data have been obtained with AMV re- verse transcriptase acting on double-stranded DNA (data not shown). Heating the template results in additional termination preceding every G in the template. Particularly obvious are the stops at positions 23 and 24. In this experiment the reac- tion mixture was incubated for an initial 10 min with 0.6 /~M dNTP's followed by an additional period as shown with 100 /~M dNTP. The lane incubated for 20 min compares most closely in total reaction time to the experiment with single- stranded template (Fig. 4). No obvious change in the pattern is seen at longer incubation times indicating that alkylation has produced true terminations rather than temporary pauses even in this double-stranded DNA.

DMS reacts largely to produce N-alkylations with the major reaction product being at the N-7 of guanine (Singer and Grunberger, 1984). In order

to see whether a more complex pattern of reaction in which O's were alkylated would have an effect, we alkylated DNA with MNNG and determined the termination pattern before and after heating. Major stops are seen at or before the sites of A in the template (Fig. 5). So for example, with pol I (Kf) stops in the unheated samples are seen at positions 19, 20, 21, 22, 25, 27, 29, 34, 35, 36, 37, 38, 39(weak), 40 and 46. Heating followed by catalysis with pol I (Kf) results in new stops at positions 23 and 24. Additional stops are also seen at positions 42, 43 and 45.

A peculiar result of heating is seen at position 27, part of the sequence ACACA, in which the bands in the heated samples seem displaced less than a full nucleotide in the 3' direction as though a different nucleotide conferring an altered electrophoretic mobility were used for termination (Rabkin and Strauss, 1984). There are interesting differences between the behavior of pol I (Kf) and

i ALl

i i i i

¸ ̧

25

2 C ~

Fig. 5. Termination of synthesis on MNNG-trea ted single-stranded templates with either E. coil pol I (Kf) or with AMV reverse transcriptase. Sequencing lanes as in Fig. 3. Number ing is from the EcoR1 site. Experimental lanes are treated with 10 m M or 30 m M MNNG. Control templates are treated with methanol. Ht, template heated I h at 65°C after M N N G treatment. Conditions are as described in Materials and Methods.

83

AMV reverse transcriptase. In all our experiments pol I produces a termination band at position 36, opposite an A and before a C which does not occur in AMV reverse transcriptase-catalyzed re- actions. Differences "higher up" in the gel are difficult to interpret since at these positions the AMV reverse transcriptase control lanes show sig- nificant termination bands, present to a lesser extent in pol I (Kf) lanes. Heating not only pro- duces new bands at some positions but results in loss of termination at others. At positions 34, 35, 40 and 46 for example, heating single-stranded templates results in loss of termination. It must also be pointed out (Fig. 5) that heating results in a diminution of AMV reverse transcriptase-cata- lyzed termination on control, non-alkylated tem- plates.

Discussion

The following points are illustrated in our ex- periments: (a) It is possible to see a regular pattern of chain termination on templates treated with simple alkylating agents. (b) Using unheated tem- plates, the terminations occur mostly before A's in the template. (c) Heating produces new bands in both DMS- and MNNG-treated templates and these bands occur mostly at or before G's in the template. (d) There are no new stops at G's in the unheated MNNG-treated samples as compared to DMS-treated templates. (d) Heating does produce a loss of stops in the single-stranded but not double-stranded templates (cf. Figs. 3 and 5 with Fig. 4). Heating also produces a loss of stops in control, non-treated DNA with AMV reverse tran- scriptase; and (e) There are differences at specific positions in the behavior of pol I (Kf) and AMV reverse transcriptase.

These observations can all be interpreted on the basis of a simple scheme. Only methyl groups at A's (and with single-stranded DNA at C's) block DNA synthesis. Neither 7-methylguanine pro- duced as the major DMS product, or O6-methyl- guanine, produced in an amount equivalent to methyl A by MNNG (Singer and Grunberger, 1983) block synthesis. When heated, the alkylated substrate depurinates and synthesis is blocked be- fore the AP sites on the template. It has previously been shown that AP sites are not always absolute

blocks, particularly to AMV reverse transcriptase (Schaaper et al., 1983) and therefore termination may either occur opposite the AP site (Sagher and Strauss, 1983) or not until the next lesion is re- ached. Some of the loss of termination sites in heated single-stranded samples may be due to the effect of heating at 65°C for 1 h in removing secondary structures which block synthesis. For example, in Fig. 5 it can be seen that heating results in the disappearance of a band at position 34. The t empla te at this po in t is: 3'(30)CTTTA(35)ACAAT(n0)AGGCG(45)AG TGT(50) TAAGG(55)5', with the underlined bases indicating a homologous region for the formation of a loop. However, this explanation does not account for the lack of termination in the un- heated control lanes unless the alkylation itself promotes changes in secondary structure.

We suppose that the blocking lesion is 3-meth- yladenine. 18% of the reaction products in double-stranded DNA are 3-methyladenine as compared to 2% for the N-1 derivative (Singer and Grunberger, 1983). The percentages are reversed in single-stranded DNA. A more compelling argu- ment is the finding that mutants devoid of 3-meth- yladenine glycosylase are very sensitive to the kill- ing action of methyl methanesulfonate, implicating this lesion in lethality (Evensen and Seeberg, 1982; Karran et al., 1982). It is generally observed that lethal agents are likely to block DNA synthesis (Painter, 1981) and to induce SOS repair (Walker, 1984). Recently Boiteux et al. (1984) have ob- tained evidence that 3-methyladenine lesions can induce SOS repair. It is certainly clear that the major reaction products on guanine are not blocks to DNA synthesis. Although the fact that such adducts are not blocks in vivo is well known (Prakash and Strauss, 1970; Lawley and Orr, 1970), these in vitro observations (see also Abbott and Saffhill, 1979) do reinforce the warning that major chemical lesions may not be the important biologi- cal products.

Acknowledgement

This work was supported by grants from the National Institutes of Health (GM 07816, CA 32436) and the U.S. Department of Energy (DE- AC02-76). KL was a trainee of a program in

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E n v i r o n m e n t a l M u t a g e n e s i s s p o n s o r e d by the N a -

t iona l C a n c e r In s t i t u t e (5T32 C A 09273). R S was

s u p p o r t e d as a t r a inee o f a p r o g r a m in In fec t i ous

D i sease s p o n s o r e d by the N I A I D (5T32 A I 07099).

W e wish to a c k n o w l e d g e the usefu l adv i ce o f Dr .

D a p h n a Sagher in the course o f these s tudies .

References

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Boiteux, S., O. Huisman and J. Laval (1984) 3-Methyladenine residues in DNA induce the SOS function sfiA in Escherichia coli, EMBO J., 3, 2569-2573

Evensen, G., and E. Seeberg (1982) Adaptation to alkylation resistance involves the induction of a DNA glycosylase, Nature (London), 296, 773-775.

Karran, P., T. Hjelmgren and T. Lindahl (1982) Induction of a DNA glycosylase for N-methylated purines is part of the adaptive response to alkylating agents, Nature (London), 296, 770-773.

Lawley, P., and D. Orr (1970) Specific excision of methylation products from DNA of Escherichia coli treated with N- methyl-N'-nitro-N-nitrosoguanidine, Chem.-Biol. Interact., 2, 154-157.

Leis, J., and J. Hurwitz (1972) Isolation and characterization of a protein that stimulates D N A synthesis from Avian myeloblastosis virus, Proc. Natl. Acad. Sci. (U.S.A.), 69, 2331-2335.

Moore, P., and B. Strauss (1979) Sites of inhibition of in vitro DNA synthesis in carcinogen- and UV-treated ~X174 DNA, Nature (London), 278, 664-666.

Moore, P., S. Rabkin and B. Strauss (1980) Termination of in vitro synthesis at AAF adducts in the DNA, Nucleic Acids Res., 8, 4473-4484.

Painter, R. (1981) Screening of mutagens by inhibition of DNA synthesis, in: E. Friedberg and P. Hanawalt (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Vol. I, Part B, Marcel Dekker, New York, pp. 569-574.

Prakash, L., and B. Strauss (1970) Repair of alkylation damage: Stability of methyl groups in Bacillus subtilus treated with methyl methanesulfonate, J. Bacteriol., 102, 760-766.

Rabkin, S., and B. Strauss (1984) A role for DNA polymerase in the specificity of nucleotide incorporation opposite N- acetyl-2-aminofluorene adducts, J. Mol. Biol., 178, 569-594.

Sagher, D., and B. Strauss (1983) Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: Uniqueness of adenine nucleotides, Biochemistry, 22, 4518-4526.

Schaaper, R., T. Kunkel and L. Loeb (1983) Infidelity of DNA synthesis associated with bypass of apurinic sites, Proc. Natl. Acad. Sci. (U.S.A.), 80, 487-491.

Singer, B., and D. Grunberger (1983) Molecular Biology of Mutagens and Carcinogens, Plenum, New York, xii + 374

PP. Strauss, B., S. Rabkin, D. Sagher and P. Moore (1982) The role

of DNA polymerase in base substitution mutagenesis on non-instructional templates, Biochemie, 64, 829-838.

Walker, G. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Micro- biol. Revs., 48, 60-93.