Genetic control of DNA repeat expansions and mutagenesis.
Shah, Kartik.
2017-04-24T15:11:27.728Z
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Abstract: Expansions of simple DNA repeats are known to cause several devastating neurodegenerative diseases in humans. Experimental systems mimicking large-scale human expansions have (1) proven difficult to develop and (2) been only moderately successful. Different cellular processes - replication, repair, homologous recombination and transcription, have been implicated in repeat instability in ... read morevarious model organisms. Only few studies so far have attempted to investigate the role of DNA polymerases. Although transcription has been implicated, an experimental system(s) that allows one to distill contributions from transcription and replication in the expansion process has not been developed. This study describes the development, optimization and utilization of genetically tractable experimental systems in the budding yeast Saccharomyes cerevisiae, to study the genetic control of repeat expansions and other repeat-associated phenomenon. By optimizing an "intronic" system that selects for large-scale expansions, the discrete length of an expansion step for Friedreich's ataxia (GAA)n repeats was determined and found to coincide with the length of an Okazaki fragment (~ 165 nts). An analysis of the effects of mutations in DNA polymerases revealed that Pol δ (delta) and Pol ε (epsilon), the leading and lagging strand polymerases, severely affect the rate of expansion. Strikingly, Pol α (alpha), the polymerase-primase that primes and dictates the size of Okazaki fragments, only affects the scale of the expansion step. Contrary to expectations, trans-lesion DNA polymerases or post-replicative PCNA modifications do not play a role. This is the first direct and distinct evidence of the involvement of replicative DNA polymerases in large-scale repeat expansions as well as the link between elementary step of expansion and Okazaki fragment size. (GAA)n repeats were found to be mutagenic, elevating the rate of point mutations in their vicinity. A term - repeat-induced mutagenesis (RIM) - was coined by us for this phenomenon. The effects of DNA polymerase mutants on RIM were strikingly similar to their effects on repeat expansions, suggesting that these two events could result from a shared replication problem. To assess the role of transcription in repeat expansions, a novel genetic system utilizing the GAL4/UAS system was developed, allowing repeats to be positioned in a non-transcribed location. Large-scale (GAA)n expansions, similar to the "intronic" system, were detected in this system, suggesting that transcriptional elongation is not required for the expansion process. In summary, this study proposes that (GAA)n repeats expand, largely irrespective of their transcriptional state, in the process of DNA replication. We believe that the replication model for expansions is probably universally true for various expandable repeats associated with human diseases. The same mechanism can also account for RIM, posing a threat to genomic integrity in individuals with long DNA repeats.
Thesis (Ph.D.)--Tufts University, 2013.
Submitted to the Dept. of Biology.
Advisor: Sergei Mirkin.
Committee: Catherine Freudenreich, Mitch McVey, Juliet Fuhrman, and Lee Zou.
Keywords: Molecular biology, and Genetics.read less - ID:
- 1c18dt397
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