Telling right from wrong: a role for DNA methylation

TIG [3]--January 1987

Telling right from wrong: a role for DNA methylation Patricia J. Pukki!a Department of Biology and Curriculum in Genetics. Universityof North Carolina. Chapel Hill, NC 27514, USA. Modified bases in the DNA of Escherichia coli were discovered initially because the thymine auxotroph 15T- accumulated high levels of an unusual base, 6-methylaminopurine, when the thymine concentration was low~'2. Although there is still no satisfactory explanation for this accumulation, the observation prompted examhtation of a variety of bacterial DHAs, which were found to contain 6-methylaminopurine at levels a tenth of those of 15T-. Marinus and Morris s took advantage of the fact that this unusual base results from postreplicative methylation of a particular subset of DNA adenine residues, and isolated mutant strains deficient in this process. Since methylation of that subset usually goes to completion, they were able to detect mutant strains on the basis of the ability of their DNA to accept methyl groups. These dam mutants were deficient in an enzyme, DNA adenine methylase, that methylates the adenine residues on both strands within GATC sequences4,s. One striking phenotype of these strains was their elevated spontaneous mutation rate e. Several other mutants with a mutator phenotype were thought to be deficient in carrying out repair of mismatched base pairs in DNA7,s. A strain deficient in mismatch repair would be expected to show a mutator phenotype only if repair usually acted to reduce replication errors. Since the consequences of random repair are equivalent to the absence of repair, a mechanism to direct the repair machinery to the newly synthesized strand was required. Wagner and Meselson9 had postulated that DNA methylation could serve such a function. At a replication fork, parental chains retain their methyl groups, while newly synthesized chains are transiently undermethylated1°. The mutator phenotype of the dam mutants suggested that the methylase was involved in a postreplicative mismatch correction process, in

which information on the undermethylated chain was preferentially removed. In the absence of methylation, repair on either chain could occur, leading to elevated error levels. Evidence that mismatch repair could indeed be directed by DNA methylation was sought using artificially constructed heteroduplexes of bacteriophage t. (Refs 11 and 12). These molecules were obtained by growing phages with different genetic markers in methylating or non-methylating hosts, separating the complementary polynucleotide chains, annealing these to produce all possible combinations of methylated and non-methylated strands, and transfecting the heteroduplex molecules (which also contained xv~smatched base pairs) into E. coil By scoring the types of phages released by each transfected cell, the frequency of mismatch repair was estimated. It was found that repair was strongly biased toward the unmcthylated chain in a heteroduplex. The bias was observed even though phage DNA is not methylated at every available GATC site under ordinary growth conditions, perhaps because packaging occurs too rapidly. The bias was enhanced when DNA that had been methylated to completion in vitro using purified DNA adenine methylase was used as the source of the methylated chains. Although hemimethylated and unmethylated heteroduplexes were repaired with similar frequencies, repair of heteroduplexes with both chains completely methylated was not detectedTM. Transfection assays and other genetic studies suggested that the products of the mutH, mutL, routs and uvrD genes also functioned in this-methyl-directed mismatch correction pathwayt2-14. The genetic experiments suggested that one or more components of the repair system recognized the mismatch, while other components of the system recognized the state of methyfation at distant GATC

~) 1987. F.Jsev~r Sdeace PulKL~.~ B-V , Amstexdam 01~,8 - 9525~7/$0 200

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sequences. Somehow the repair machinery was permitted to act if an unmethylated GATC was present, and directed to remove information located on the unmethylated chain. Key advances in elucidating the mechanisms involved in this 'action at a distance' have come from in vitro studies. Of prime importance in this work was the decision by Lu, Clark and Modrich to monitor the complete repa~ reaction, rather than hypothetical intermediates (such as molecules nicked at mismatches) TM. Repair synthesis at mismatched EcoRI restriction endonuclease recognition sites was observed in cell-flee extracts by virtue of the restoration of an EcoRl-sensitive site. The repair reactions were shown to be dependent on the presence of a mismatch, the presence of unmethylated GATC sequences (which directed the reaction to the unmethylated chain) and products of the mutlt. mulL, routs and uvrD genes. In vitro complementation of mutant extracts has provided the means of purifTing these componunts and reconstituting an efficient mismatch repair system in vitro. Two recent observations are of particular interest. Su and ModrichTM have purified avd characterized the routs product. This 97kDa protein was observed to bind to regions of DNA that contained mismatched base pairs. Each of four mismatches that were studied showed a different affinity for the routs protein. This binding was not dependent on the presence of unn~ethylated GATC sequences. An unexpected finding was that the DNA sequence protected from DNAse I digestion by the protein was not centered over the mismatch. Furthermore, distinct protection patterns, or 'footprints', were obtained for two different mismatches at an identical location. Understanding how this protein interacts with DNA and other components of the repair system should provide key insights into the biological significance of altered DNA conformations. A second recent advance was the demonstration that unmethylated or hemimethylated GATC sequences are required for mismatch repair. This requirement has been demonstrated both in ViVO17 and in V~O TM. These and previous in vitro experiments ~s.19 have demonstrated that correction of the mismatch is associated with extensive repair syn- t...,'k

t chonical tips

• on the unmethylated strand. This synthesis requires the presence of a mismatch as well as a hemimethylated or unmethylated GATC sequence. Among the substrates studied in vitro were two 6 kb phage DNAs which differed at only a single nucleotide, such that only one had a GATC sequence. The consequences of this single nucleotide change were striking: extensive mismatch correction and repair synthesis occurred only on the substrate that contained a GATC sequence. Thus, molecules lacking GATC sequences and molecules with fully rnethylated GATC sequences are not repaired by the m u t t t L S pathway. Although other correction systems may also operate ~°.21, the strong mutator phenotype exhibited by strains with lesions in this pathway argues that this system is of prime importance in

TIG [3] --]anuary 1987 in eukaryotes. Using a limited series I of mismatched substrates based on DNA from the SV40 virus, Hare and Taylor 2s observed ~ influence of methylation on marker recovery. In a particularly striking experiment, the addition of two methyl groups to one strand of a heteroduplex resulted in a dramatic increase in recovery of that genotype after transfection into CV-I cells. In addition to its considerable theoretical significance, the methyldirected mismatch correction system in E . coil has proved to be of practical importance. An understanding of the properties of this system has permitted optimization of oligonucleotide-directed mutagenesis zs'~7. Reconstitution of the complete system in vitro using purified components is awaited with interest.

vivo.

Since eukaryotes generally lack methyl adenine, it has been proposed that analogous repair systems might R e f e r e n c e s utilize asymmetries at the replication ' I Dunn, D, B. and Smith, J.D. (1955) Nature 175, 336-337 fork, such as nicks 2~-, to achieve 2 Dunn, D. B. and Smith, J.D. (1958) correct orientation. Alternatively, Biochem. ]. 68, 627--636 both mismatched strands might be 3 Marinus, M. G. and Morris, N. IL (1973) replaced with information on the ]. Bacteriol. 114, 1143-1150 other newly replicated chrorna 4 Lacks, S. A. and Greenberg, B. (1977) ]. Mol. Biol. 114, 153-168 tid ='7"4. A third possibility is that G. E. and Modnch, P. (1979) 5-methyl cytosine might direct mis- 5 J.Geier, Biol. Chem. 254, 1408--1413 match correction, as this is the 6 Bale, A•, d'Alarcao, M. and Marinus, primary post-synthetic modification M. G. (1979) Murat. Res. 59, 157-165

7 Nevers, P. and Spatz, H• (1975)Mol. Gen. Genet. 139, 233-243 8 Rydberg, B. (1978)Murat. Res. 52, 11-24

9 Wagner, R. and Meselson, M. (1976) Prec. Nell Acad. Sci. USA 73, 413,5--4139 10 Lyons, S. M. and Schendel, P. F. (1984) ]. Badedoi. 159, 421-423 11 Radman, M., Wagner, R, E., Glickman, B.W. and Meselson, M. (1980) in Progress in Environmentcl Mutagenesis (M. Abcevic, ed•), pp. 121-130, Elsevier 12 Pukldla, P. J., Peterson, J., Herman, G., Modrich, P. and Meselson, M. (1983) Genetics 104, 571-582 13 Gl~ckman,B. W• and Radman, M. (1980) Prec. NatlAcad~ Sd. USA 77, 1063-1067 14 McGraw,B. R. and Marinus, M. G. (1980) Mol• Gen. Genet. 178, 309-316 15 Lu, A-L., Clark, S. andModrich,P. (1983) Proc. NatlAcad. Sd. USA 80, 4639-4643 16 Su, S-S. and Modrich,P. (1986)Prec. Nell Aced• Sd. USA 83, 5057-5061 17 Laengle-Rouault, F., Maenhaut-Michel, G. and Radman, M. (1986)EMBO]. 5,

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18 Lahue, R. S., Su, S-S. and Mndrich, P. Prec. Nell Acad. Sd. USA (in press) 19 Lu. A-L. et ai. (1984) Cold Spdng Harbor Syrup. Quant. Biol. 49, 589-596 20 Fishel, R. A., Siegel, E. C. and Kolodner. R. (1986)]. MoL Biol. 188, 147-157 21 Mannarelb, B. M. et el. (1985)Prec. Natl Acad. ScL USA 82, 4468-4472 22 Ahmnd,A., Holloman,W. K. and Holliday, R. (1975) Nature 258, 54--56 23 Hastings, P. J. (1984)Cold Spying Harbor Syrup. Ouant. Biol. 49, 49-53 24 Wagner, R. et aL (1984) Cold Spdv~ Harbor Syrup• Quant. Biol. 49, 611-615 25 Hare, J. T. and Taylor,J. H. (1985)Prec. Natl Acad. Sci. USA 82, 7350-7354 26 Kramer, W., Schughart, K. and Fritz, H-J. (1982) Nucleic Adds Res. 10, 6475--6485 27 Kramer, W. et al. (1984)Nucleic Adds

Res. 12, 9441-9456

Creation of a restriction site at a pre-determined position in a DNA sequence

RNA detection with singlestranded probes

Experiments with recombinant DNA often require the insertion or deletion of specific DNA fragments. However, the target sequence does not always contain a suitable restriction site. A common solution to this problem is to use an exonuclease, BAL 31, to generate a series of progressively shorter DNA fragments, from which the fragment containing the exposed target sequence can be identified by a laborious selection procedure. The specificity of the selection procedure can be improved by ligation of a

The definitive determination of coding strands and mapping of RNA-coding regions can be achieved with a simple twostage hybridization procedure. First, singie-stranded M13 DNA conraining the DNA fragment of interest is hybridized to RNA gel blots, The blots are then probed with isotopicallylabelled M13 RF DNA. The purity of the single-stranded probe allows definitive assignment of coding strands and also increases the sensitivity of the technique, since competing strands are absent. The first hybridization increases the target size for the second (which is specific for M13 sequences), thus further enhancing sensitivity. Many probes for various regions along a large DNA fragment can be tested simultaneously but only one radioactive probe, the M13 RF, need be prepared, thus reducing cost and exposure to radioactivity.

Selection oligomer

Fspl

EcoRI

............. TGC GCAGAATTCTGC Target sequence L,nker

specially designed ollgomer to the BAL 31 fragments. This oligomer contains one complete restriction site (to facilitate insertion of the ligation product into a plasmid) and half of a different restriction site (Fig. 1). The second site is completed only when ligated to the original target sequence. After an initial screening by in sifu hybridizationto an oligo-probe specific for the target DNA-linker junction, the identity o[ the recombinunt clone containing the target sequence can be confirmed by cutting with the restriction nuclease specific for the newly for[ned site at the linker-target DNA junction. The availability of a suitable restriction enzyme is the only major constraint on the number of different restri~on sites that can be generated at a s p e ~ c target site• Kalyan, N• K., Hung, P. P., Levner, M. H.,

Fig. 1• Ligelion o/ a spedfic oligomerto theBAL 31 Dheer, S. K. and Lee, S. G. (1986)Gene 42, frasme~t containing the ta~et DNA sequonce• 331--337

Boyex,P. D. (1986)NuddcAcidsRes• 14, 7505

© 1987.Else~erSeencel~bbshersB.V, Amsterdam0~.68- 9525/87/$0.200