POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Pol theta in replication repair. DNA polymerase theta is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining of DNA double-strand breaks. How full-length human DNA polymerase theta performs microhomology-mediated end-joining and is regulated by the helicase and disordered central domain remains unknown. We find that the helicase upregulates DNA polymerase theta microhomology-mediated end-joining activity in an ATPase-independent manner. Using single-particle microscopy, we find that DNA polymerase theta forms large multimeric complexes that promote DNA accumulation and end-joining. We further find that the disordered central domain regulates DNA polymerase theta multimerization and governs its DNA substrate requirements for end-joining. In summary, these studies identify major regulatory functions for the helicase and central domains in DNA end-joining and the structural organization of DNA polymerase theta. / Biomedical Sciences
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/3374 |
| Date | January 2019 |
| Creators | Ozdemir, Ahmet Yunus |
| Contributors | Pomerantz, Richard, Graña-Amat, Xavier, Skorski, Tomasz, Tempera, Italo, Mazin, Alexander |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 157 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/3356, Theses and Dissertations |
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