A two‐dimensional ¹H‐NMR study of the dam methylase site: comparison between the hemimethylated GATC sequence, its unmethylated analogue and a hemimethylated CATG sequence

Abstract

We report two-dimensional NOE (NOESY) spectra on the sequence d(GCGATCATGG) d(CCATGATCGC) which contains the unmethylated dam site. As expected the DNA adopts a B-form conformation but appears to be distorted at the TG step of the second strand. This distorsion, probably bending, is not seen on the opposite strand. When the first strand is methylated on adenine in the GATC or CATG sequence the NOESY spectra indicate little or no change in the conformation. However the single strand-duplex exchange is slowed down to the slow-exchange region on a proton NMR time scale. We have assigned the exchangeable imino and cytidine amino resonances of the three duplexes. From the imino linewidths as a function of temperature, we observe that the unmethylated and the hemimethylated Gm6ATC duplexes melt normally from the ends. However, this is not so for the hemimethylated Cm6ATG duplex which, apart from the terminal base pairs, melts cooperatively and at higher temperature. In spectra recorded in H 2 0 a second duplex is observed, for the Gm6ATC sequence, which we have not been able to identify. It is however unlikely to be a hairpin structure. Ultravioletmelting curves also indicate the presence of two transitions for this duplex.The effect of methylation upon base-pair lifetimes has been studied by comparing the above three duplexes. Little effect is observed upon methylation in the GATC sequence but a drastic increase in the lifetimes of all base pairs is observed upon methylation in the CATG sequence.DNA methylation has been proposed as a means of strand discrimination for mismatch repair [l]. An adenine methylase, dam methylase, specific for the sequence GATC has been studied [2]. dam methylase acts primarily on hemimethylated sequences [3] and the methylation lags somewhat behind replication such that newly synthesized strands are transiently undermethylated [4, 51. Full methylation of GATC sequences appears to inhibit mismatch repair completely [3, 6, 71.In previous studies [8 -101 on sequences methylated on both strands we observed that helix formation was drastically slowed down relative to the unmethylated sequences. Also methylation considerably increases the lifetime of the A . T base pair in the closed state [ll]. On the other hand, the helix conformation was not observed to be changed by methylation.While most of the adenine methylases known today act on sequences with a purine 5' to the adenine, one methylase, PstI acts on a sequence with a pyrimidine in the 5' direction [12]. Here we report results on three non-selfcomplementary decamers: one is hemimethylated in the GATC sequence, a second in the CATG sequence and these are compared with the unmethylated sequence. MATERIALS AND METHODSThe decanucleotides were synthesized by a modified phosphate triester and phosphoamidite method on silica gel support [13].The duplexes, Duplex I 5'd(G1 C2 G3 A4 T5 c 6 A7 T8 G9 Glo) strand A 3'd(C2oG19 C18 T17 A16 GIs T14 A13 C12 C1d strand B Duplex11 5'd(G1 Cz G3 A4 T5 c 6 m6A7T8 G9 Glo strandA 3'd(CzoG19 C18 T17 A16 G l 5 T14...

Structure and Dynamics of Hemimethylated GATC Sites

“…5A). These findings support previous findings of base pair lifetimes in GATC sites, even though the previously adopted methods differ from ours (15).…”

“…1A), and there is no significant conformational difference between methylated DNA and unmethylated DNA with the same nucleotide sequence. These studies, however, demonstrated that the adenine N 6 hemimethylated DNA has a lower melting temperature and distinct dynamic characteristics represented by slow duplexsingle strand exchange (13)(14)(15)(16). Here, we investigate the solution structure and dynamics of two dodecamer DNA duplexes that contain either a single hemimethylated or unmethylated GATC site.…”

N6-Methyladenine: A Conserved and Dynamic DNA Mark

“…6mA within GATC sequences causes slight DNA unwinding of 0.5°/methyl group (Cheng et al 1985). Consistent with these observations, two-dimensional NMR studies suggest that, in almost all cases, 6mA has only minor effects on helix conformation, as it retains the canonical B-form (Fazakerley et al 1985; Quignard et al 1985; Fazakerley et al 1987). 6mA occurring directly after thymines, on the other hand, causes severe unwinding and bending of the DNA helix relative to the canonical B conformation (Fazakerley et al 1989).…”

“…The effects of 6mA on the thermodynamic stability and folding of DNA appear to be sequence-specific (Fazakerley et al 1987). Indeed, when 6mA occurs directly after a T this can cause a highly altered structure that is overwound and bent (Fazakerley et al 1989).…”

Dam methylase fromEscherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates

“…The other goal of this study was to determine whether or not one or the other strand was significantly influenced by these substitutions and thus a preference of one over the other strand could be detected. (46)(47)(48). Substitutions in the sequences studied gave irregular responses.…”