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Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome
1Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143.
Abstract:
We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.
Insights
Blunt-end DNA double-strand breaks from restriction enzymes can cause mutations and complex rearrangements in mammalian cells. This study reveals how these breaks lead to illegitimate recombination and mutagenesis.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions.
- Understanding DSB repair mechanisms is crucial for comprehending mutagenesis and genome stability.
Purpose of the Study:
- To investigate mutations induced by blunt-end DSBs in the adenine phosphoribosyl-transferase (APRT) gene.
- To analyze the DNA sequence alterations occurring during the repair of restriction enzyme-induced breaks.
Main Methods:
- Electroporation of restriction endonucleases (PvuII, EcoRV, StuI) into Chinese hamster ovary (CHO-AT3-2) cells.
- Culturing and selection of mutant colonies.
- DNA sequence analysis of APRT gene mutations.
- Southern blot analysis for complex genomic rearrangements.
Main Results:
- Restriction enzyme-induced mutations included small deletions (1-36 bp) and insertions at cleavage sites.
- Many deletions involved overlapping complementary bases at recombination junctions.
- Complex mutations, including translocations and inversions, were detected via Southern blot.
Conclusions:
- Blunt-end DSBs can induce illegitimate (nonhomologous) recombination in mammalian chromosomes.
- These breaks play a significant role in mutagenesis, contributing to genomic instability.