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Assay for Human Rad51-Mediated DNA Displacement Loop Formation

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同源重组（Homologus Recombination) 是指发生在姐妹染色单体（sister chromatin) 之间或同一染色体上含有同源序列的DNA分

同源重组（Homologus Recombination) 是指发生在姐妹染色单体（sister chromatin) 之间或同一染色体上含有同源序列的DNA分子之间或分子之内的重新组合。同源重组需要一系列的蛋白质催化，如原核生物细胞内的RecA、RecBCD、RecF、RecO、RecR等；以及真核生物细胞内的Rad51、Mre11-Rad50等等。

在人类细胞中，同源重组由Rad51介导，在有ATP的条件下，Rad51聚合在单链DNA上，形成一个核蛋白丝，通常是被称为presynaptic filament。presynaptic filament定位在同源染色体双联体DNA分子上，并且起催化作用促进双联体形成DNA置换环，又称为D-loop。

该实验手册就是介绍在体外检测D-loop的方法，用一个放射标记的ssDNA（单链DNA）寡核苷酸和一个未标记的同源染色体超螺旋DNA做底物，用琼脂糖胶电泳进行检测。为了加强检测效果，添加辅助因子（Hop-2-Mnd复合物或Rad54。这一实验方案为研究者提供了一个剖析同源重组机制的研究路径。

Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5120

Assay for Human Rad51-Mediated DNA Displacement Loop Formation Steven Raynard and Patrick Sung1

Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA

1 Corresponding author

INTRODUCTION

Homologous recombination is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome metabolism and maintenance. The homologous recombination reaction is mediated by the Rad51 recombinase. In the presence of ATP, Rad51 polymerizes on single-stranded DNA (ssDNA) to form a nucleoprotein filament that is commonly referred to as the "presynaptic filament." The presynaptic filament is capable of locating a homologous duplex DNA molecule and catalyzing invasion of the duplex to form a DNA displacement loop called the "D-loop." This protocol describes an in vitro D-loop assay that uses a radiolabeled ssDNA oligonucleotide and a nonlabeled homologous supercoiled duplex DNA as substrates, and agarose gel electrophoresis together with PhosphorImaging for product analysis. To enhance the efficiency of the D-loop reaction, an ancillary factor (the Hop2-Mnd1 complex or Rad54) is included in the reaction. This reconstituted system provides researchers a biochemical means to dissect the mechanisms of the homologous recombination machinery.

MATERIALS

Reagents

It is imperative that highly purified proteins are used to avoid artifacts arising from contaminating nuclease, DNA helicase, or topoisomerase activities. For optimal activity of the purified homologous recombination proteins, avoid repeated freeze-thaw cycles.

[γ-32 P]ATP (10 mCi/mL, 6000 Ci/mmoL; Amersham Bioscience)

Agarose gel (0.9%)

Agarose gel loading buffer

D-loop reaction buffer (5X)

Oligonucleotide D1 (5'-AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT-3')

The oligonucleotide is complementary to positions 1932-2022 of pBluescript SK DNA.

pBluescript SK plasmid DNA (Stratagene)

Plasmid Midi kit (QIAGEN)

Proteinase K (Roche Applied Science)

Recombinant Hop2-Mnd1 complex, purified (5 µM) or recombinant human Rad54, purified (5 µM) (see Step 5)

Purify Hop2-Mnd1 complex as described by Chi et al. (2007) .

Purify Rad54 as described by Sigurdsson et al. (2002) .