Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair
| Autor: | Alice Witsell, Amy Baker, Robin L. Armstrong, Orlando D. Schärer, Nikolai Renedo, Kelly Beagan, Upasana Roy, Mitch McVey |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Adenosine Triphosphatase
0301 basic medicine Cancer Research DNA End-Joining Repair DNA polymerase DNA-Directed DNA Polymerase Biochemistry Polymerases DNA annealing DNA polymerase delta 0302 clinical medicine Catalytic Domain Drosophila Proteins Genetics (clinical) Polymerase DNA clamp biology Drosophila Melanogaster Physics Animal Models Enzymes Cell biology Insects Nucleic acids Physical sciences Experimental Organism Systems 030220 oncology & carcinogenesis Nucleic acid thermodynamics Drosophila DNA polymerase mu Research Article lcsh:QH426-470 Arthropoda Nucleic acid synthesis DNA polymerase II Annealing (genetics) Biophysics DNA repair Research and Analysis Methods 03 medical and health sciences Model Organisms DNA-binding proteins Genetics Animals Chemical synthesis Molecular Biology Ecology Evolution Behavior and Systematics Biology and life sciences DNA synthesis Phosphatases Organisms Proteins DNA Processivity Invertebrates Molecular biology lcsh:Genetics Biosynthetic techniques DNA Repair Enzymes 030104 developmental biology Enzymology biology.protein Microhomology-Mediated End Joining Nucleotide excision repair |
| Zdroj: | PLoS Genetics PLoS Genetics, Vol 13, Iss 5, p e1006813 (2017) |
| ISSN: | 1553-7404 |
| DOI: | 10.1371/journal.pgen.1006813 |
| Popis: | Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance. Author summary Error-prone DNA Polymerase θ (Pol θ) plays a conserved role in a mutagenic DNA double-strand break repair mechanism called microhomology-mediated end joining (MMEJ). In many organisms, it also participates in a process crucial to the removal/repair of DNA interstrand crosslinks. The exact mechanism by which Pol θ promotes these processes is unclear, but a clue may lie in its dual-domain structure. While the role of its polymerase domain has been well-studied, the function of its helicase-like domain remains an open question. Here we report an absolute requirement for ATPase activity of the helicase-like domain during interstrand crosslink repair in Drosophila melanogaster. We also find that although end joining frequency does not decrease in ATPase-dead mutants, ATPase activity is critical for generating templated insertions. Using purified Pol θ protein, we show that it can bypass synthetic substrates mimicking interstrand crosslink intermediates and can promote MMEJ-like reactions with partial double-stranded and single-stranded DNA. Together, these data demonstrate a novel function for the helicase-like domain of Pol θ in both interstrand crosslink repair and MMEJ and provide insight into why the dual-domain structure has been conserved throughout evolution. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|