Chapter 37
Sequence-Specific Methylation of Single- and Double-Stranded DNA by Methylnitrosourea
Downloaded via UNIV OF ARIZONA on July 25, 2018 at 07:58:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
R. W. Wurdeman and B. Gold Eppley Institute and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198
DNA damage induced by chemical and physical agents and "spontaneous" processes can lead to base pair mismatches and bulge sites (1). These non-Watson -Crickbase pairing motifs may be converted into somatic mutations during DNA repair or replication. The impact of these abnormal base pairing arrangements on DNA structure was probed using the sequence selective methylation of DNA at N7 -G by N-methyl-N-nitrosourea (MNU) (2). The reactions of MNU with 5'-[ ]P -labeledsingle-stranded (s-s) DNA 1 [1, 5'-d(CACTG G G ACTG C)], complementary double-stranded (d-s) DNA 1+2 [2, 3'-d(GTGACCCTGACG)], d-s DNA's with a G-G mismatch (1+3, [3, 3'-GTGACGCTGACG]), d-s DNA with a G-A mismatch (1+4 [4, 3'-GTGACACTGACG]), d-s DNA with a G-T mismatch (1+5 [5, 3'-GTGACTCTGACG]), d-s DNA 1+6 (6, 3'-GTGAC_CTGACG) with a single bulged G and d-s DNA 1+7 (7, 3'-GTGA_C_TGACG) with two bulged G's are reported. In addition, the effect of temperature on the methylation of 1+2 is reported along with the T 's of all the duplexes and the CD spectra of 1, 2 and 1+2. 32
5
6
7
11
m
Experimental Oligomers 1-7 were prepared on a DNA synthesizer and purified by LC. 1 was S'-pPJ-end-labeled with T4 kinase and purified by electrophoresis on a 2% polyacrylamide gel. The S'-^PJ-end-labeled 1 (with or without 2-7) was incubated with 500 μΜ MNU in 10 mM buffer (pH 7.8) containing 100 mM NaCl and 1 mM EDTA for 2 h at 20 °C. The DNA was treated with piperidine to selectively convert the N7-MeG lesions into strand breaks (3) and the DNA was run on 20% polyacrylamide denaturing gel. The gel then was exposed to X-ray film and the autoradiogram analyzed on a scanning densitometer (Fig. 1). The effect of temperature on the methylation of 1+2 was performed as described above except for the change in temperature (Fig. 2). The denaturation of 1+2-7 as a function of temperature was followed by monitoring their UV absorbance at 260 nm: T of 1+2 (56 °C), 1+2 (44 °C), 1+4 (51 °C), l + £ (46 °C), 1+6 (43 °C) and 1+7 (31 °C). The CD spectra of 168 μΜ 1, 2 and 1+2 are shown in Fig. 3. m
0097-6156/94/0553-0346$08.00/0 © 1994 American Chemical Society
Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
37.
WURDEMAN & GOLD
υ ο
\ ο ΙΟ
1 + 1
υ ο ο ο ο ο ΟΙΟ ου α3
