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PMID: 11381128 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 98 ·No. 12 ·2001-06-05 ·Pages 6742-6

Ellis HM, Yu D, DiTizio T, Court DL

Abstract

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 lambda 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.

MeSH Terms
Amino Acid Sequence Base Sequence DNA Repair DNA Replication DNA, Single-Stranded/metabolism DNA-Binding Proteins/physiology Molecular Sequence Data Mutagenesis Rec A Recombinases/analysis Recombination, Genetic Viral Proteins
Chemicals
DNA, Single-Stranded DNA-Binding Proteins Viral Proteins beta protein, Bacteriophage lambda Rec A Recombinases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Ellis H M
Gene Regulation and Chromosome Biology Laboratory, Division of Basic Sciences, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702, USA.
Yu D
DiTizio T
Court D L
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.121164898
Published
2001-06-05
Epub
2001-00-29
Pages
6742-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC34423
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.121164898 PMC Full Text View journal details
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