Genetic control of DNA repeat expansions and mutagenesis.
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... read morein various
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
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