High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

被引:416
作者
Ellis, HM [1 ]
Yu, DG [1 ]
DiTizio, T [1 ]
Court, DL [1 ]
机构
[1] NCI, Gene Regulat & Chromosome Biol Lab, Div Basic Sci, Ft Detrick, MD 21702 USA
关键词
D O I
10.1073/pnas.121164898
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:6742 / 6746
页数:5
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