Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

First Advisor's Name

Yuan Liu

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Alejandro Barbieri

Second Advisor's Committee Title

committee member

Third Advisor's Name

Yuk-Ching Tse-Dinh

Third Advisor's Committee Title

Committeee member

Fourth Advisor's Name

Xiaotang Wang

Fourth Advisor's Committee Title

Committee member

Keywords

DNA damage, DNA repair, BER, FEN1, FAN1, R-loop, TNR

Date of Defense

3-26-2020

Abstract

The human genome is constantly attacked by endogenous and exogenous sources of DNA damage that generates DNA base lesions and strand breaks leading to genome instability, cell death, and diseases. To combat these adverse effects, cells have evolved a robust DNA repair mechanism called “the DNA base excision repair (BER),” which efficiently removes DNA lesions maintaining genome stability. However, its underlying molecular mechanisms remain to be elucidated. In my dissertation research, I explored the molecular mechanism by which BER modulates trinucleotide repeats (TNR) by processing non-B form structures such as hairpins and R-loops through the coordination among BER enzymes and cofactors and the proteins from other DNA repair pathways. For the first time, we discovered that Fanconi anemia-associated nuclease 1 (FAN1) coordinated with a key BER enzyme, flap endonuclease 1 (FEN1), to prevent repeat expansion by promoting CAG repeat deletion during BER of oxidative DNA damage. We further demonstrated that the BER cofactor, PCNA/ubPCNA, coordinated with FAN1 to attenuate CAG repeat expansion during BER in a CAG repeat hairpin. Exploring the mechanisms by which BER regulates TNR instability by processing bulky non-B form structures, R-loops. We found that an abasic lesion on the non-template strand of a CAG repeat R-loop was incised by AP endonuclease 1 (APE1), converting the R-loop into a double-flap intermediate containing an RNA:DNA hybrid. This inhibited DNA polymerase β (pol β) DNA synthesis and stimulated FEN1 cleavage of the repeats leading to repeat deletion. We showed that FEN1 partially cleaved the RNA strand on a CAG repeat R-loop to facilitate pol β skip-over of a hairpin leading to repeat deletion. We further identified a new role of FEN1 of resolving R-loops through the BER pathway. We found that FEN1 efficiently cleaved RNA during the processing of DNA lagging strand and R-loops and demonstrated that FEN1 also used its flap tracking to track down to the DNA region to make a cleavage removing the RNA leading to the lagging strand maturation and R-loop resolution via BER. Our study provides new insights into the molecular mechanism by which BER maintains genome stability and integrity.

Identifier

FIDC008956

ORCID

https://orcid.org/0000-0002-2535-2630

Previously Published In

Beaver, J. M., Lai, Y., Xu, M., Casin, A. H., Laverde, E. E., & Liu, Y. (2015). AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair. Nucleic Acids Research, 43(12), 5948–5960.

Jiang, Z., Xu, M., Lai, Y., Laverde, E. E., Terzidis, M. A., Masi, A., Chatgilialoglu, C., Liu, Y. (2015). Bypass of a 5′,8-cyclopurine-2′-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks. DNA Repair, 33, 24–34. Masi A, Sabbia A,

Ferreri C, Manoli F, Lai Y, Laverde E.E., Liu Y, Krokidis MG, Chatgilialoglu C, Faraone MR. (2019) Diastereomeric Recognition of 5’,8-cyclo-2’- Deoxyadenosine Lesions by Human Poly(ADP-ribose) Polymerase 1 in a Biomimetic Model. Cells, 8, 116.

Laverde, E.E, Lai, Y., and Liu, Y.; Proliferating Cell Nuclear Antigen Stimulates Flap Endonuclease 1 to Modulate GAA Repeat Stability Via Base Excision Repair. Poster presented at the 46th Annual Meeting of Environmental Mutagenesis and Genomics Society (EMGS), Sep. 26th-30th, 2015, New Orleans, LA. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS Volume: 56 Supplement: 1 Pages: S62-S62 Meeting Abstract: P22 Published: AUG 2015 138

Laverde, E.E, Lai, Y., and Liu, Y.; Base Excision Repair in a GAA Repeat R-loop Leads to GAA Repeats Instability. Poster presented at the 48th Annual Meeting of Environmental Mutagenesis and Genomics Society (EMGS), Sep. 9th-13th, 2017, Raleigh, NC. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS Volume: 58 Supplement: 1 Pages: S57-S57 Meeting Abstract: P10 Published: SEP 2017

Laverde, E.E, Lai, Y., and Liu, Y.; R-loop at GAA Repeats Mediates Repeat Instability Through Modulation of BER Enzymatic Activity. Poster presented at the 49th Annual Meeting of Environmental Mutagenesis and Genomics Society (EMGS), Sep. 22th-26th, 2018, San Antonio, TX. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS Volume: 59 Supplement: 1 Pages: 88- 88 Meeting Abstract: P17 Published: SEP 2018

Ren Y, Lai Y, Laverde E.E., Lei R, Rein HL, Liu Y. (2017). Modulation of trinucleotide repeat instability by DNA polymerase β polymorphic variant R137Q. PLoS ONE 12(5): e0177299.

Suzol SH, Howlader AH, Wen Z, Ren Y, Laverde E.E., Garcia C, Liu Y, and Wnuk SF. (2018). Pyrimidine Nucleosides with a Reactive (β-Chlorovinyl)sulfone or (β- Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA. ACS Omega 3(4), 4276-4288.

Wen Z, Tuttle P, Howlader H, Vasilyeva A, Gonzalez L, Tangar A, Lei R, Laverde E.E, Liu Y, Miksovska J, and Wnuk SF. (2019). Fluorescent 5-Pyrimidine and 8- Purine Nucleosides Modified with an N-Unsubstituted 1,2,3-Triazol-4-yl Moiety. The Journal of Organic Chemistry, 84 (6), 3624-3631

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