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Titolo:
Importance of terminal base pair hydrogen-bonding in 3 '-end proofreading by the Klenow fragment of DNA polymerase I
Autore:
Morales, JC; Kool, ET;
Indirizzi:
Univ Rochester, Dept Chem, Rochester, NY 14627 USA Univ Rochester Rochester NY USA 14627 Dept Chem, Rochester, NY 14627 USA Univ Rochester, Dept Biochem & Biophys, Rochester, NY 14627 USA Univ Rochester Rochester NY USA 14627 & Biophys, Rochester, NY 14627 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 10, volume: 39, anno: 2000,
pagine: 2626 - 2632
SICI:
0006-2960(20000314)39:10<2626:IOTBPH>2.0.ZU;2-D
Fonte:
ISI
Lingua:
ENG
Soggetto:
ESCHERICHIA-COLI; SPONTANEOUS MUTATION; DUPLEX DNA; EXONUCLEASE; REPLICATION; FIDELITY; MECHANISM; BACTERIOPHAGE-T4; DIFLUOROTOLUENE; EXCISION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
33
Recensione:
Indirizzi per estratti:
Indirizzo: Kool, ET Stanford Univ, Dept Chem, Stanford, CA 94305 USA Stanford Univ Stanford CA USA 94305 Chem, Stanford, CA 94305 USA
Citazione:
J.C. Morales e E.T. Kool, "Importance of terminal base pair hydrogen-bonding in 3 '-end proofreading by the Klenow fragment of DNA polymerase I", BIOCHEM, 39(10), 2000, pp. 2626-2632

Abstract

We describe studies aimed at evaluating the physical factors governing therate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene(F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T.Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited, Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues atthe duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.

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Documento generato il 03/04/20 alle ore 12:33:51