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Modulation of DNA end joining by nuclear proteins
Li Liang1, Li Deng, Yanping Chen
1Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, USA. liang@biology.rutgers.edu
The Journal of Biological Chemistry
|July 14, 2005
Summary
Microhomology-mediated end joining (MHEJ), a mutagenic DNA repair pathway, is favored by high DNA-to-protein ratios in cell extracts. Factors like Ku and histone H1 inhibit MHEJ, while flap endonuclease 1 promotes it.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks are repaired by homologous recombination and non-homologous end joining (NHEJ).
- NHEJ can be error-free or mutagenic.
- Microhomology-mediated end joining (MHEJ) repairs breaks using flanking microhomologous sequences and is typically mutagenic.
Purpose of the Study:
- To investigate factors influencing the relative contribution of MHEJ to DNA end joining.
- To characterize the role of nuclear proteins and DNA/protein ratios in modulating MHEJ.
- To establish a cell-free system for evaluating MHEJ modulation.
Main Methods:
- Utilized cell-free nuclear extracts from mammalian cells.
- Employed DNA substrates with 10-bp microhomologous repeats at the ends.
- Assessed MHEJ occurrence under varying DNA/protein ratios and with specific protein depletions/additions.
Main Results:
- MHEJ predominated over error-free joining at high DNA/protein ratios.
- Ku and histone H1 were identified as inhibitors of MHEJ.
- Flap endonuclease 1 promoted MHEJ, while intervening sequences between microhomologies reduced its efficiency.
Conclusions:
- The relative abundance of nuclear proteins significantly impacts the choice between error-free and MHEJ pathways.
- Specific proteins like Ku, histone H1, and flap endonuclease 1 play critical roles in regulating MHEJ.
- The developed cell-free assay is a valuable tool for studying DNA end-joining mechanisms and identifying modulators.