Abstract
Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.
Highlights
((GA)n)as models of homopurine-homopyrimidine S1- to as (GA),) appear frequently in SHS withheterogeneous hypersensitive sites, we show that cleavageof the al- sequence (4, 5 ),while (GA), homopolymeric SHS are associternate DNA structure by S1 nuclease ated with several eukaryotic genes, encoding sea urchin hisis length-dependent,inbothsupercoiled andlinear tones [7], Drosophila histones (81, Drosophila heat shock forms, whichare similar because of the identityof their proteins [9], mouse renins (IO),and human U1 RNA [11].A nicking profiles
The length of flanking se- (GA), element is present in the 3’ flanking region of the quences, the presenceof borders, and theDNA topol- mouse Cp heavychain gene, while a (CT), element is present bppDot uogeaNrnriyerAiemshhaawefeanfnlvieidttecBcdhsatit-rflhDaeferevNedrepeAieanneq.luatcuTtctohi,lhnleabiefobtouBtrtrtiihmdufteheomaaertrmileottbpwenoeersfo(nathwt(apeGutetheenAeorsnis)ott3)prn.8tuhohhEcomaextdsuotiaereaunesls1ttsee0iror.in4nsb-abaetaecxalsks-oe-s t r upatricoenrng-oedixttoeh(hniTeinsbCeopic)ftu,oe,rtnnihadfhireaeeidnnmatcrfleoorlruonacsmcoeotnfDiMtvCaridioHtnys(eCo1t dhp2ah)Ei.tinlPmTahtachgrpueeesnleatseutmttrrh(eaa1delc3ltec)sseo.ktlfIlnns(oTtwewCr5hne)i’,sc,ft,hfrilna(asGggntmliAkymie),n,un,ag,t heteronomous, in agreement withthe nicking patterns lates transcription binds to a (GA)ll stretch present in the generated by S 1 a n d mungbean nucleases and by spacer region between the Drosophila histone genes H3 and venom phosphodiesterase
Primers were from New England Biolabs; S1 nuclease, T4 polynucleotide kinase, and NACS.52 resin were from Bethesda Research Laboratories; mung bean nuclease, EcoRI linkers, and phosphorylatedaandS( referred to as AG and CT deca
Summary
1 and 2, mers) were from P-L Biochemicals; bacterial alkaline phosphatase, venom phosphodiesterase, and DNase I werefrom Worthington; reverse transcriptase was from Life Sciences; Dimethyl sulfate was from Aldrich DEP was from Sigma; 7-methyl-dGTP, synthesized as and Ref. 3 fora review). They are presentonnaked described [15],was a generous gift from P. Srinivasan; [a-32P]dNTPs supercoiled (but usually not relaxed or linear) DNA (1, 4,5 ) Such S1-hypersensitive sites (SHS’), which may play a role in transcription or chromatin organization, reside in homopurine-homopyrimidinestretches[4,5],and,despitetheir sensitivity t o single strand-specific nucleases, they seem to be (700 Ci/mmol) and[T-~’P]ATP (3,000 Ci/mmol) werefromNew England Nuclear. Presumably arising from a spontaneous deletion, which gave a weakly positive C-testto mpCT38, was shown by sequencing to contain 19 GA doublets (mpGA19)
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