Johnson A W, Kolodner R D
Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
J Biol Chem. 1994 Feb 4;269(5):3664-72.
Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA from single-stranded and homologous linear duplex DNA. The initial pairing reaction requires limited exonucleolytic digestion of the double-stranded DNA (dsDNA) by the intrinsic 5' to 3' exonuclease of Sep1 or by an exogenous exonuclease. Subsequent strand exchange proceeds without the need for exonuclease activity. Sep1 degrades linear dsDNA at a rate of 20 nucleotides/min with an average processivity of 45 nucleotides. During strand exchange reactions joint molecules are first observed after 1-2 min, suggesting that only limited digestion is necessary for pairing. The linear dsDNA was found to pair with single-stranded DNA (ssDNA) when it was resected by only 22 nucleotides, and linear dsDNA digested by more than 22 nucleotides was observed only in joint molecules. Approximately 20 nucleotides were also the minimum extent of digestion that could support pairing. In the absence of exonuclease activity, Sep1-promoted pairing requires dsDNA molecules with single-stranded tails homologous to the circular ssDNA. These results suggest that Sep1-promoted strand exchange requires a single-strand annealing event prior to the strand displacement phase of the reaction.
来自酿酒酵母的链交换蛋白1（Sep1）催化单链和同源线性双链DNA形成异源双链DNA。初始配对反应需要Sep1的固有5'至3'核酸外切酶或外源核酸外切酶对双链DNA（dsDNA）进行有限的核酸外切消化。随后的链交换无需核酸外切酶活性即可进行。Sep1以每分钟20个核苷酸的速度降解线性dsDNA，平均持续合成能力为45个核苷酸。在链交换反应中，1-2分钟后首次观察到连接分子，这表明配对仅需要有限的消化。当线性dsDNA仅被切除22个核苷酸时，发现其与单链DNA（ssDNA）配对，并且仅在连接分子中观察到被消化超过22个核苷酸的线性dsDNA。大约20个核苷酸也是能够支持配对的最小消化程度。在没有核酸外切酶活性的情况下，Sep1促进的配对需要具有与环状ssDNA同源的单链尾巴的dsDNA分子。这些结果表明，Sep1促进的链交换在反应的链置换阶段之前需要单链退火事件。
