Publication Date

2023

Document Type

Dissertation

Committee Members

Michael Leffak, Ph.D. (Advisor); Courtney Sulentic, Ph.D. (Committee Member); Michael Kemp, Ph.D. (Committee Member); Weiwen Long, Ph.D. (Committee Member); Yong-jie Xu, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Faithful DNA replication is essential to maintain genomic integrity. Compromises in this process are among the prime causes of cancer and other genetic disorders. Among several factors that can interfere with error-free DNA replication, the formation of non-B DNA structures by microsatellite sequences is critical. Microsatellites are 1-9 bp long tandem repetitive regions that can form secondary structures like hairpins, triplex, Gquadruplex etc. and undergo expansions due to replication fork stalling to cause several diseases. Hairpin forming microsatellites have been shown to induce DNA DSBs and lead to genetic instability and mutagenesis. The goal of this study is to understand the effects on genome stability of triplex and G quadruplex secondary structures formed by the homopurine-homopyrimidine (Pu/Py)88 microsatellite sequence. We hypothesize that this sequence can induce microsatellite instability, genetic instability, and mutagenesis through break induced replication (BIR). Our study shows that the (Pu/Py)88 sequence generates DNA DSBs, is unstable in a replication polarity dependent manner and shows a high frequency of mutagenesis. The pattern of mutagenesis suggests that BIR may function to repair replication- dependent DNA DSBs. To investigate whether triplex or G quadruplex structures are responsible for this instability, we generated cell lines that can specifically form either of these structures. Our data shows that both structures are unstable, but the G quadruplexes are more deleterious between the two. To determine other reasons leading to mutagenesis we studied the ability of triplex and G quadruplexes to form extrachromosomal DNA (eccDNA). Our data reveals that the non-B DNA structures can generate eccDNA which cause elevated mutagenesis and gross chromosomal rearrangements. Since our data suggests BIR functioning in the cells for DNA repair, we focused on understanding the role of fork remodeling proteins that operate in BIR like Rad51 and Mus81. Our data indicate that depending on the non-B DNA structure formed the Rad51 requirements are different. Similarly, Mus81, which induces DNA DSBs can function in both S and G2/M phase of the cell cycle based on the non-B DNA structure formed. Overall, our study reveals the potential contribution of the (Pu/Py) microsatellite sequence to genetic instability.

Page Count

150

Department or Program

Biomedical Sciences

Year Degree Awarded

2023

Download


Share

COinS