Synthesis-Dependent Microhomology-Mediated End Joining: A Unifying Model For Multiple DNA Double Strand Break Repair Outcomes.
Yu, Amy.
2011
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Abstract: DNA double-strand breaks (DSBs) are repaired by homology-directed
repair or non-homologous end joining. End-joining repair can be classified as classical
non-homologous end joining, which requires DNA ligase 4, or "alternative" end joining
(alt-EJ), which does not. The mechanism of alt-EJ is largely uncharacterized. We show that
Drosophila melanogaster DNA polymerase theta (pol theta), ... read moreencoded by the mus308 gene, plays
a role in alt-EJ. In pol theta mutants, end joining is impaired, creating large deletions.
Sequencing suggests that pol theta promotes the use of long microhomologies during alt-EJ.
These results suggest a mechanistic link between alt-EJ and interstrand crosslink repair.
Alt-EJ repair frequently correlates with junctional microhomology but also produces
junctions without microhomology, and the role of microhomology in alt-EJ remains unclear.
To investigate this, we examined inaccurate repair of an I-SceI DSB with few nearby
microhomologies in Drosophila. Lig4 deficiency did not affect junctional microhomology, but
significantly increased insertions. Many insertions appeared templated. Based on sequence
analysis, we propose a model of synthesis-dependent microhomology-mediated end joining
(SD-MMEJ), in which de novo synthesis by an accurate non-processive DNA polymerase creates
microhomology. Repair junctions with apparent blunt joins, junctional microhomologies, and
short indels (deletion with insertion) are often considered to reflect different repair
mechanisms. However, a majority of each type had structures consistent with the predictions
of our SD-MMEJ model, suggesting that a single underlying mechanism could be responsible
for all three. SD-MMEJ is Ku70, Lig4, and Rad51-independent, but impaired in mus308 (POLQ)
mutants. Although the SD-MMEJ model is a powerful conceptual tool, the question remained
whether or not patterns associated with SD-MMEJ would be found at other DSBs. A
meta-analysis of end-joining repair junctions produced in the course of previous studies in
Drosophila showed that substantial numbers of the junction sequences had SD-MMEJ consistent
repeats. Patterns of repeats varied in a sequence specific manner consistent with the
predictions of the SD-MMEJ model. Correlating the patterns of SD-MMEJ consistent repeats at
breaks from C-NHEJ proficient and deficient backgrounds with sequence content and break end
structure confirms a complex relationship among sequence content, genetic background, and
end-joining repair outcome.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Biology.
Advisor: Mitch McVey.
Committee: Susan Ernst, Catherine Freudenreich, Sergei Mirkin, and Maria Jasin.
Keywords: Biology, Genetics, and Molecular Biology.read less - ID:
- 5m60r3373
- Component ID:
- tufts:21061
- To Cite:
- TARC Citation Guide EndNote
