Expansions of simple DNA repeats are implicated in nearly thirty hereditary disorders in humans. This proposal concentrates on molecular mechanisms responsible for repeat expansions. During the previously funded period we have found that replication forks stalled at expandable repeats in mammalian cells. The length of the repeat, which caused replication inhibition, closely matched the threshold length for its expansion in human pedigrees. We have further studied the control of repeat-mediated replication blockage in yeast to discover that the fork stabilizing proteins, Tof1 and Mrc1, facilitated replication through expandable repeats. Most significantly, we have developed a new experimental system to analyze large-scale repeat expansions in yeast. The unique advantage of this system is that it allowed us to monitor expansions of the carrier-size repeats well into the disease-size range. Expansion rates were strongly elevated upon inactivation of the replication fork stabilizer, Tof1, while significantly decreased in the lack of the DNA helicase Sgs1 or the post-replicative repair regulator Rad6. Altogether these data implicate DNA replication and/or post-replicative repair in repeat instability;we will further assess this hypothesis in yeast and mammalian cells in this proposal. We will study large-scale expansions of various tri-, tetra-and pentanucleotide repeats in our yeast experimental system by analyzing the rates of repeat expansions and visualizing the replication fork progression through various repeats. Expanded repeats inhibited gene expression in yeast as they do in human diseases. We will, therefore, analyze the mechanisms responsible for gene repression in our system by studying the effects of various repeats on transcription, RNA splicing and RNA stability. For repeats that are unstable when transcribed, we will develop a different system for their large-scale expansions, so that they are positioned in a non- transcribed area. To get insight into the genetic control of repeat instability, we will analyze repeat expansions and contractions in our large collection of mutants affecting DNA replication, repair and recombination in yeast. We will further perform genetic screens for yeast mutants that show either an increased rate of large-scale repeat expansions or a decreased rate of repeat contractions using gene disruption with a mutagenized yeast genomic library. In mammalian cells, we will evaluate replication of various expandable repeats in the pSV2neo episome using two-dimensional electrophoresis of replication intermediates. We will also study whether expandable repeats trigger episomal fragility. Expression of mammalian homologues of the genes, which came up from the yeast screens, will be knocked down by siRNAs to study their role in repeat-mediated replication blockage and fragility. Finally, we will attempt to develop a new system for monitoring large-scale repeat expansions in human cells using a specifically designed HyTK selectable cassette combined with the Flip-In integration approach. The long-term goal of this proposal is to understand molecular mechanisms responsible for repeat expansions and contractions in humans.

Public Health Relevance

More than two dozen human hereditary diseases are caused by uncontrollable expansions of simple DNA repetitions within human genes. They include debilitating neurological disorders, such as Huntington's disease, fragile X mental retardation, myotonic dystrophy and others. This proposal is to unravel molecular mechanisms responsible for this phenomenon.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060987-10
Application #
7804544
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Hagan, Ann A
Project Start
2001-04-01
Project End
2013-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
10
Fiscal Year
2010
Total Cost
$355,670
Indirect Cost
Name
Tufts University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
073134835
City
Medford
State
MA
Country
United States
Zip Code
02155
Kim, Jane C; Harris, Samantha T; Dinter, Teresa et al. (2017) The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats. Nat Struct Mol Biol 24:55-60
Neil, Alexander J; Kim, Jane C; Mirkin, Sergei M (2017) Precarious maintenance of simple DNA repeats in eukaryotes. Bioessays 39:
Tsutakawa, Susan E; Thompson, Mark J; Arvai, Andrew S et al. (2017) Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability. Nat Commun 8:15855
McGinty, Ryan J; Rubinstein, Rachel G; Neil, Alexander J et al. (2017) Nanopore sequencing of complex genomic rearrangements in yeast reveals mechanisms of repeat-mediated double-strand break repair. Genome Res 27:2072-2082
McGinty, Ryan J; Puleo, Franco; Aksenova, Anna Y et al. (2017) A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA)n Repeats Associated with Friedreich's Ataxia. Cell Rep 20:2490-2500
Kim, Jane C; Mirkin, Sergei M (2015) Putting the Brakes on Huntington Disease in a Mouse Experimental Model. PLoS Genet 11:e1005409
Aksenova, Anna Y; Han, Gil; Shishkin, Alexander A et al. (2015) Expansion of Interstitial Telomeric Sequences in Yeast. Cell Rep 13:1545-51
Shah, Kartik A; Mirkin, Sergei M (2015) The hidden side of unstable DNA repeats: Mutagenesis at a distance. DNA Repair (Amst) 32:106-12
Shah, Kartik A; McGinty, Ryan J; Egorova, Vera I et al. (2014) Coupling transcriptional state to large-scale repeat expansions in yeast. Cell Rep 9:1594-602
Mirkin, Ekaterina V; Mirkin, Sergei M (2014) To switch or not to switch: at the origin of repeat expansion disease. Mol Cell 53:1-3

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