FORMATION OF DNA TRIPLEXES ACCOUNTS FOR ARRESTS OF DNA-SYNTHESIS AT D(TC)N AND D(GA)N TRACTS

被引:131
作者
BARAN, N [1 ]
LAPIDOT, A [1 ]
MANOR, H [1 ]
机构
[1] TECHNION ISRAEL INST TECHNOL,DEPT BIOL,IL-32000 HAIFA,ISRAEL
关键词
HOOGSTEEN HYDROGEN BONDS; TRIPLE HELICES; POLYPURINES AND POLYPYRIMIDINES; DNA REPLICATION; GENE AMPLIFICATION;
D O I
10.1073/pnas.88.2.507
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
To study the mechanism of arrest of DNA synthesis at d(TC)n and d(GA)n sequences, single-stranded DNA molecules including d(TC)27 or d(TC)31 tracts or a d(GA)27 tract were used as templates for in vitro assays of complementary DNA synthesis performed by extension of a primer with the Klenow polymerase or the Taq polymerase (Thermus aquaticus DNA polymerase). Electrophoresis of the products revealed that arrests occurred around the middle of these tracts. The arrests in the d(TC)n sequences were eliminated when dATP or dGTP was replaced with the analogue 7-deaza dATP or 7-deaza dGTP, respectively, or when the templates were preincubated with the Escherichia coli singlestrand binding protein (SSB). Preincubation of the template including a d(GA)27 tract with SSB has also eliminated the arrests at this sequence. Furthermore, arrests did not occur at d[G(7-deaza A)]27 or d[(7-deaza G)]27 tracts when molecules including such tracts were used as templates. These results are compatible with the notion that the arrests were caused by formation of d(TC)i.d(GA)i.d(TC)i and d(GA)i.d(GA)i.d(TC)i triplexes, in which the bases in the uncopied portions of the d(TC)n tracts, or of the d(GA)27 tract, and the purine bases in the newly synthesized d(TC)i.d(GA)i duplexes were bound by hydrogen bonds. In the assays performed with the Taq polymerase, the pH dependence (in the range of 6.0-9.0) and the temperature dependence of the arrests were determined. As the pH was lowered, the arrests in the d(TC)27 tract were enhanced, in line with the expected properties of d(TC)i.d(GA)i.d(TC)(i)triplexes. The arrests in the d(GA)27 tract were enhanced by an increase in the pH. At pH 7.2 the arrests in the d(GA)27 tract persisted up to 80-degrees-C, whereas the arrests in the d(TC)27 tract were eliminated at 50-degrees-C; these results presumably reflect the relative stabilities of the two triplexes mentioned above at this physiological pH value and could be biologically significant.
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页码:507 / 511
页数:5
相关论文
共 36 条
[1]   UNUSUAL SEQUENCE ELEMENT FOUND AT THE END OF AN AMPLICON [J].
BARAN, N ;
LAPIDOT, A ;
MANOR, H .
MOLECULAR AND CELLULAR BIOLOGY, 1987, 7 (07) :2636-2640
[2]   ONION SKIN REPLICATION OF INTEGRATED POLYOMA-VIRUS DNA AND FLANKING SEQUENCES IN POLYOMA-TRANSFORMED RAT-CELLS - TERMINATION WITHIN A SPECIFIC CELLULAR DNA SEGMENT [J].
BARAN, N ;
NEER, A ;
MANOR, H .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1983, 80 (01) :105-109
[3]   STRUCTURAL POLYMORPHISM OF HOMOPURINE HOMOPYRIMIDINE SEQUENCES - THE SECONDARY DNA-STRUCTURE ADOPTED BY A D(GA.CT)22 SEQUENCE IN THE PRESENCE OF ZINC IONS [J].
BERNUES, J ;
BELTRAN, R ;
CASASNOVAS, JM ;
AZORIN, F .
EMBO JOURNAL, 1989, 8 (07) :2087-2094
[4]   POLYNUCLEOTIDES .16. FORMATION OF THE TRIPLE-STRANDED POLYNUCLEOTIDE HELIX, POLY(A-A-U) [J].
BROITMAN, SL ;
IM, DD ;
FRESCO, JR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (15) :5120-5124
[5]   INTRAMOLECULAR DNA TRIPLEXES, BENT DNA AND DNA UNWINDING ELEMENTS IN THE INITIATION REGION OF AN AMPLIFIED DIHYDROFOLATE-REDUCTASE REPLICON [J].
CADDLE, MS ;
LUSSIER, RH ;
HEINTZ, NH .
JOURNAL OF MOLECULAR BIOLOGY, 1990, 211 (01) :19-33
[6]   STRUCTURE OF GENES FOR MEMBRANE AND SECRETED MURINE IGD HEAVY-CHAINS [J].
CHENG, HL ;
BLATTNER, FR ;
FITZMAURICE, L ;
MUSHINSKI, JF ;
TUCKER, PW .
NATURE, 1982, 296 (5856) :410-415
[7]   ORGANIZATION OF THE RAT GAMMA-FIBRINOGEN GENE - ALTERNATIVE MESSENGER-RNA SPLICE PATTERNS PRODUCE THE GAMMA-A AND GAMMA-B (GAMMA') CHAINS OF FIBRINOGEN [J].
CRABTREE, GR ;
KANT, JA .
CELL, 1982, 31 (01) :159-166
[8]   PHYSICAL AND CHEMICAL PROPERTIES OF NUCLEIC ACIDS [J].
FELSENFE.G ;
MILES, HT .
ANNUAL REVIEW OF BIOCHEMISTRY, 1967, 36 :407-&
[9]  
FOGEL M, 1969, VIROLOGY, V40, P174
[10]   SEQUENCE-SPECIFIC RECOGNITION OF THE MAJOR GROOVE OF DNA BY OLIGODEOXYNUCLEOTIDES VIA TRIPLE HELIX FORMATION - FOOTPRINTING STUDIES [J].
FRANCOIS, JC ;
SAISONBEHMOARAS, T ;
HELENE, C .
NUCLEIC ACIDS RESEARCH, 1988, 16 (24) :11431-11440