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A Study of Mutagenesis by Translesion Synthesis DNA Polymerases Using A Novel High-throughput Mutation Assay System

Chen, Yizhang
http://orcid.org/0000-0001-7330-1187
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1534928845629071

Abstract Details

2018, Doctor of Philosophy (PhD), Ohio University, Biological Sciences (Arts and Sciences).
DNA mutations can have devastating effects on cell growth, development, proliferation and survival through the regulation of gene expression and protein activity. Within various processes that can create mutations, DNA damage is the major source of mutations. During the lifetime of a cell, DNA is constantly attacked by DNA damaging agents which can be either endogenously produced from cell metabolism or from surrounding environment. In addition, the intrinsic instability of DNA structure challenges genome integrity. While multiple DNA damage repair pathways can be activated to remove damage and restore DNA structure, a damage tolerance process, known as translesion synthesis (TLS), is essential for maintaining genome stability. TLS is a process in which a special group of DNA polymerases (TLS polymerases) synthesize DNA over a DNA lesion without repairing it. Since the DNA lesion cannot be the guide for the accurate base-pairing during DNA synthesis, TLS is often mutagenic and considered as the major mechanism for DNA damage-induced mutagenesis. In this study, a newly developed mutation assay system that takes the advantage of next-generation sequencing (NGS) technology, a high-throughput and efficient DNA sequencing technology, that was used to investigate the mutagenesis by TLS in vitro. This in vitro system successfully evaluated i) the fidelity of DNA synthesis by yeast TLS polymerases Pol Eta and Pol Zeta on intact templates; ii) base mis-incorporation generated by TLS over a specific DNA lesion (AP site) by multiple yeast DNA polymerases; iii) mis-incorporations generated through TLS on templates with random DNA lesions that were created by a DNA damaging agent (cisplatin). Yeast TLS polymerases mainly incorporated A and G opposite damaged sites. Mutations created by different TLS polymerases showed distinct signatures on the same template which could help in distinguishing their functions during TLS. Furthermore, while TLS introduced mutations at the lesion, the presence of DNA damage affected TLS fidelity around the lesion.
Tomohiko Sugiyama (Advisor)
231 p.
Biology
DNA Damage; Translesion Synthesis; Mutagenesis; Next-generation Sequencing; Pol Eta; Pol Zeta

Recommended Citations

Citations

  • Chen, Y. (2018). A Study of Mutagenesis by Translesion Synthesis DNA Polymerases Using A Novel High-throughput Mutation Assay System [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1534928845629071

    APA Style (7th edition)

  • Chen, Yizhang. A Study of Mutagenesis by Translesion Synthesis DNA Polymerases Using A Novel High-throughput Mutation Assay System. 2018. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1534928845629071.

    MLA Style (8th edition)

  • Chen, Yizhang. "A Study of Mutagenesis by Translesion Synthesis DNA Polymerases Using A Novel High-throughput Mutation Assay System." Doctoral dissertation, Ohio University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1534928845629071

    Chicago Manual of Style (17th edition)

ohiou1534928845629071
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© 2018, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.