Unscheduled Replication at Microsatellite Repeats
Open Access
- Author:
- Teo, Yee Voan
- Area of Honors:
- Microbiology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Maria Krasilnikova, Thesis Supervisor
Dr. Sarah Ellen Ades, Thesis Honors Advisor
Richard John Frisque, Faculty Reader - Keywords:
- Microsatellite
Unscheduled replication
non-B DNA structures - Abstract:
- Friedreich’s ataxia is an autosomal recessive neurodegenerative disorder that is primarily caused by the expansion of GAA trinucleotide repeats. This expansion was found to occur during the first pre-embryonic cell division, when the chromatin is not fully mature. The presence of GAA repeats was found to increase the efficiency of unscheduled replication in mammalian cells. Due to its repetitive nature, the GAA repeat can potentially form triplexes that are accompanied by single-stranded DNA regions, which can then serve as loading docks for replication initiation protein. In order to determine the molecular mechanism of the unscheduled replication initiation at the GAA repeats, we used a plasmid with GAA57 repeats as a model system to determine which replication initiation protein would bind to the GAA repeats when the chromatin is not fully assembled. We observed the recruitment of three replication initiation proteins—DNA polymerase α, origin recognition complex (ORC) and mini chromosome maintenance (MCM) to the DNA in the proximity of the GAA repeat during early embryogenesis, which supports the hypothesis that unscheduled replication occurs at the GAA repeat in the absence of a complete chromatin structure. Short regions of DNA that are synthesized from this unscheduled replication are expelled from the genome in the form of linear DNA fragments and induce recombination with other genomic regions, which could then lead to genomic instability. In addition, we also showed that tetranucleotide repeats that form triplex structures stall the unscheduled replication in length dependent manner, which is indicative of the same stalling mechanism. We also studied the progression of mammalian replication fork through several tetranucleotides in the first and subsequent replication cycles. We observed that tetranucleotides that could potentially form triplex structures significantly stalled the first replication cycle whereas tetranucleotides that might form hairpin structures stalled subsequent replication cycles. Lastly, we also compared the strength of replication stalling with the relative mutability of different tetranucleotides at orthologous locations in human and chimpanzee, which showed that stalling intensity is motif and length dependent.