Open AccessProcessing of DNA Prior to Illegitimate Recombination in Mouse Cells
Author(s) -
Gordon W. Henderson,
Paul Simons
Publication year - 1997
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.17.7.3779
Subject(s) - biology , homologous recombination , exonuclease , heteroduplex , recombination , dna , exonuclease iii , microbiology and biotechnology , genetics , non homologous end joining , restriction enzyme , homologous chromosome , in vitro recombination , non allelic homologous recombination , d loop , dna repair , genetic recombination , gene , dna polymerase , molecular cloning , mitochondrial dna , complementary dna , escherichia coli
In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 5'-->3' exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 3'-->5' exonuclease processing also occurs.