High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
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DOI：
10.1073/pnas.121164898
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发表时间：
2001-06-05
影响因子：
11.1
通讯作者：
Court, DL
中科院分区：
文献类型：
--
作者：
Ellis, HM;Yu, DG;Court, DL
同源DNA重组是生物体内一个基本的再生过程。然而，在大多数生物体中，同源重组是一种罕见的事件，需要一套复杂的反应和广泛的同源性，我们在本文中证明，噬菌体A的β蛋白产生重组体的染色体DNA使用合成的单链DNA（ssDNA）短至30个碱基长。这种ssDNA重组可用于诱变或修复染色体，其效率在处理的细胞中产生高达6%的重组体。从机制上讲，β蛋白，一种Rad52样蛋白，结合并退火ssDNA供体到DNA复制叉附近的互补单链以产生重组体。这种与ssDNA的同源重组为研究和修饰从细菌病原体到真核生物的基因组提供了新的途径。β蛋白和ssDNA可能被证明普遍适用于许多生物体中的DNA修复。
Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology, We demonstrate in this paper that Beta protein of phage A generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant, This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes, Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.