Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering.
Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering.
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DOI：
10.1016/j.jmb.2011.01.030
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发表时间：
2011-03-18
影响因子：
5.6
通讯作者：
Court DL
中科院分区：
文献类型：
--
作者：
Sawitzke JA;Costantino N;Li XT;Thomason LC;Bubunenko M;Court C;Court DL
在大肠杆菌中与单链DNA寡核苷酸重组是一种高效、快速的体内修饰复制子的方法。通过寡核苷酸重组产生的核苷酸改变为研究细胞过程提供了新的分析方法。单链核酸外切酶抑制寡核苷酸重组，而通过突变所有已知的四种核酸外切酶，重组增加。增加寡聚浓度或添加非特定载体的寡聚滴定可滴定核酸外切酶。在一个寡核苷酸重组的模型中，DNABeta蛋白在复制分叉处使寡核苷酸退火为互补的单链λ。错配被产生，甲基定向错配修复(MMR)系统作用于消除抑制重组的错配。通过寡核苷酸设计避免MMR的三种方法除了期望的改变外，还包括1)从该改变开始的C~C失配6个BP，2)四个或更多相邻失配，或3)在四个或更多连续摆动位置的失配。后者被证明对进行高频改变很有用，这种改变只改变目标氨基酸序列，甚至允许修改必要的基因。DNA的有效摄取对于寡核苷酸介导的重组非常重要。对寡聚或质粒的摄取依赖于生长介质，在最低限度的培养基中生长的细胞比在丰富的培养基中生长的细胞减少10,000倍。利用重组工程的全基因组工程技术将受益于优化的重组频率和更好地理解DNA复制和细胞分裂等生物过程如何影响在多个染色体位点形成的重组体。这里描述了单个细胞中多个位点的重组事件。
Recombination with single-strand DNA oligonucleotides (oligos) in E. coli is an efficient and rapid way to modify replicons in vivo. The generation of a nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and by mutating all four known exonucleases recombination is increased. Increasing the oligo concentration or addition of non-specific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change 1) a C~C mismatch six bp from that change, 2) four or more adjacent mismatches, or 3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high frequency changes that alter only the target amino-acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is growth media-dependent and is 10,000-fold reduced for cells grown in minimal vs rich medium. Genome-wide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.
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DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
14.9
作者：
Muyrers, JPP;Zhang, YM;Stewart, AF
通讯作者：
Stewart, AF
影响因子：
2.9
作者：
Joshi, A;Rao, BJ
通讯作者：
Rao, BJ
影响因子：
3.5
作者：
Mythili, E;Kumar, KA;Muniyappa, K
通讯作者：
Muniyappa, K
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL