11月16日，DNA Repair在線發(fā)表了北京生命科學(xué)研究所研究員杜立林實(shí)驗(yàn)室的最新研究成果,，首次報(bào)道了一個(gè)在裂殖酵母中檢測染色體上進(jìn)行的末端連接修復(fù)的方法,，并利用此方法分析了機(jī)理不同的三類末端連接修復(fù)方式,。
非同源末端連接（NHEJ）是哺乳動(dòng)物中修復(fù)DNA雙鏈斷裂的主要方式。參與NHEJ的修復(fù)蛋白如Ku,，ligase IV,，XLF，和X家族DNA聚合酶在裂殖酵母中都是保守的,。通過表達(dá)一個(gè)高特異性的核酸內(nèi)切酶來產(chǎn)生基因組上一個(gè)特定位置的雙鏈斷裂,，作者建立了結(jié)合傳統(tǒng)測序和高通量測序全面分析不精確修復(fù)產(chǎn)物序列的方法，對突變體中修復(fù)產(chǎn)物序列的分析驗(yàn)證了Ku,，ligase IV和XLF在經(jīng)典NHEJ中的核心作用,，以及X家族DNA聚合酶Pol4在間隙填補(bǔ)(Gap Filling)中的重要作用。研究發(fā)現(xiàn)在Ku或ligase IV缺失的突變體中,，兩種非NHEJ的另類末端連接仍能發(fā)生,。其中一種方式利用了DNA斷裂位置兩側(cè)的微同源（microhomology）序列并造成幾百至幾千堿基的缺失，另外一種方式則只在酶切位點(diǎn)處引入堿基置換,。
本文的實(shí)驗(yàn)結(jié)果主要由北京大學(xué),、清華大學(xué)和北京生命科學(xué)研究所聯(lián)合培養(yǎng)博士研究生項(xiàng)目（PTN-BBS）2011級(jí)研究生李鵬在輪轉(zhuǎn)期間完成，其他貢獻(xiàn)者有研究生李俊,，李明,，竇坤，和生物信息分析員張美俊和索芳,。此項(xiàng)研究由科技部和北京市資助,，在北京生命科學(xué)研究所完成。（生物谷Bioon.com）
>>延伸閱讀：Science：腫瘤抑制基因在DNA修復(fù)上也起關(guān)鍵作用
>>延伸閱讀：Nature：癌細(xì)胞的DNA修復(fù)并非單行道
>>延伸閱讀：Cell：華人科學(xué)家發(fā)現(xiàn)重構(gòu)DNA修復(fù)系統(tǒng)
>>延伸閱讀：Science：DNA修復(fù)細(xì)胞反應(yīng)圖譜完成
>>延伸閱讀：DNA Repair：浙大黃俊等解析DNA修復(fù)機(jī)制
>>延伸閱讀：DNA Repair：RAD18蛋白在DNA修復(fù)中的作用
doi:10.1016/j.ndarep.2011.10.011
PMC:
PMID:
Multiple end joining mechanisms repair a chromosomal DNA break in fission yeast
Li P, Li J, Li M, Dou K, Zhang MJ, Suo F, Du LL.
Non-homologous end joining (NHEJ) is an important mechanism for repairing DNA double-strand breaks (DSBs). The fission yeast Schizosaccharomyces pombe has a conserved set of NHEJ factors including Ku, DNA ligase IV, Xlf1, and Pol4. Their roles in chromosomal DSB repair have not been directly characterized before. Here we used HO endonuclease to create a specific chromosomal DSB in fission yeast and examined the imprecise end joining events allowing cells to survive the continuous expression of HO. Our analysis showed that cell survival was significantly reduced in mutants defective for Ku, ligase IV, or Xlf1. Using Sanger sequencing and Illumina sequencing, we have characterized in depth the repair junction sequences in HO survivors. In wild type cells the majority of repair events were one-nucleotide insertions dependent on Ku, ligase IV, and Pol4. Our data suggest that fission yeast Pol4 is important for gap filling during NHEJ repair and can extend primers in the absence of terminal base pairing with the templates. In Ku and ligase IV mutants, the survivors mainly resulted from two types of alternative end joining events: one used microhomology flanking the HO site to delete sequences of hundreds to thousands of base pairs, the other rejoined the break using the HO-generated overhangs but also introduced one- or two-nucleotide base substitutions. The chromosomal repair assay we describe here should provide a useful tool for further exploration of the end joining repair mechanisms in fission yeast.
