A FLUORESCENCE-BASED ASSAY FOR MONITORING HELICASE ACTIVITY

被引：123

作者：

RANEY, KD

SOWERS, LC

MILLAR, DP

BENKOVIC, SJ

机构：

[1] PENN STATE UNIV, DEPT CHEM, UNIV PK, PA 16802 USA

[2] CITY HOPE NATL MED CTR, DIV PEDIAT, DUARTE, CA 91010 USA

[3] SCRIPPS RES INST, DEPT MOLEC BIOL, LA JOLLA, CA 92037 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1994年 / 91卷 / 14期

关键词：

DDA HELICASE; STOPPED FLOW SPECTROSCOPY; 2-AMINOPURINE;

D O I：

10.1073/pnas.91.14.6644

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：

A continuous fluorescence-based assay is described for measuring helicase-mediated unwinding of duplex DNA. The assay utilizes an oligonucleotide substrate containing the fluorescent adenine analog, 2-aminopurine, at regular intervals. 2-Aminopurine forms a Watson-Crick type base pair with thymine and does not distort normal B-form DNA. Fluorescence of the 2-aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this substrate by the T4 dda helicase restores the fluorescence of the 2-aminopurines and is easily followed using stopped-now or steady-state fluorescence spectroscopy. The fluorescence-based assay provides rate data comparable to that obtained from conventional discontinuous assays using labeled substrates and additionally furnishes a means for following a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.

引用

页码：6644 / 6648

页数：5

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