Publication Date

2023

Document Type

Dissertation

Committee Members

Michael Leffak, Ph.D. (Advisor); Michael Kemp, Ph.D. (Committee Member); Thomas Brown, Ph.D. (Committee Member); Shulin Ju, Ph.D. (Committee Member); Michael Markey, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Microsatellites are short tandem repeats of DNA (1-9 nucleotides) that are unstable in the genome. They can expand and contract which leads to mutations and rearrangements that jeopardize genomic integrity. When encountered by a replication fork, repeats of these microsatellites can form non-B DNA structures which cause replication forks to stall and collapse eventually leading to double stranded breaks (DSBs) and replication stress. One form of repair of replication dependent DSBs happens via break induced replication (BIR), a highly mutagenic repair pathway. The main objective of this study is to understand how ATTCT pentanucleotide repeats affect replication and chromosomal instability through quantitively measuring replication dependent DSBs at these microsatellites and by investigating recombination signatures generated at these microsatellites after repair. Expansion of ATTCT pentanucleotide repeats at the ataxin 10 locus is responsible for the human disease spinocerebellar ataxia type 10 (SCA10). Therefore, we focus on three aims. Aim 1: To determine the effect of expanded ATTCT repeats on genome stability when functionally replacing the DNA unwinding element (DUE) of the c-myc origin of replication. In a novel dual fluorescence reporter gene system, we use flow cytometry, inverse PCR, DNA sequencing to monitor BIR mutagenesis of the expanded ATTCT repeat and flanking DNA. In the same aim, we also investigate the role of the replication stabilizing proteins STN1, COPS2 and Pol η in preventing BIR at ATTCT microsatellites. This was done by siRNA knockdown of the proteins of interest, flow cytometry to monitor changes in flow profile after knockdown and sequencing inverse PCR products to detect mutagenesis signatures. Aim 2: To determine the effect of different patient-derived ATTCT repeats (SCA10 variants) and variations in these repeats’ sequences on genome stability. Aim 3: To determine the effect of ATTCT repeats and variations in these repeats’ sequences on extrachromosomal circular DNA generation and their effect on genome stability. Aims 2 and 3 have been achieved using flow cytometry and inverse PCR sequencing.

Page Count

179

Department or Program

Biomedical Sciences

Year Degree Awarded

2023

ORCID ID

0000-0001-7846-9974

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