Break-induced replication: A review and an example in budding yeast
Break-induced replication: A review and an example in budding yeast
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DOI：
10.1073/pnas.151008198
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发表时间：
2001-07-17
影响因子：
11.1
通讯作者：
Haber, JE
中科院分区：
文献类型：
--
作者：
Kraus, E;Leung, WY;Haber, JE
断裂诱导复制(BIR)是一种非互惠重组依赖的复制过程，是修复断裂染色体的有效机制。我们回顾了BIR在维持基因组完整性中扮演的关键角色，包括在断裂的复制叉处重新启动DNA复制，以及在没有端粒酶的情况下维持端粒。以往的研究表明，基因打靶不是通过线性化转化片段的末端与目标染色体之间的简单交叉来实现的，而是涉及类似于BIR的广泛的新DNA合成。我们在酿酒酵母中检测了基因打靶，其中转化的DNA只有一端与染色体序列同源。将含有着丝粒的线性化着丝粒DNA和LEU2基因的5‘端转化为一株由ADE1取代了LEU2基因5’端的菌株，防止了简单的同源基因置换成为Leu2(+)。回收了Ade1(+)Leu2+转化子，其中发现了完整的LEU2基因和长达7kb的附加序列，并连接了具有非同源末端连接(NHEJ)特征的微同源性。在其他实验中，用缺乏ARS的DNA片段转化细胞，这些DNA片段只与URA3基因末端添加的50个碱基对的ADEZ同源。恢复了自主复制的环，包含URA3和从染色体DNA复制的长达8kb的ADEZ相邻序列，包括附近的ARS，因此，线性化DNA片段的末端可以通过BIR启动新的DNA合成，其中新合成的DNA被置换，随后由NHEJ形成环。
Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5 ' end of the LEU2 gene at one end was transformed into a strain in which the 5 ' end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2(+). Ade1(+) Leu2+ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADEZ added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADEZ-adjacent sequences, including a nearby ARS, copied from chromosomal DNA, Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.