title: Altered replication in human cells promotes DMPK (CTG)(n) · (CAG)(n) repeat instability.
abstract: Myotonic dystrophy type 1 (DM1) is associated with expansion of (CTG)(n) · (CAG)(n) trinucleotide repeats (TNRs) in the 3' untranslated region (UTR) of the DMPK gene. Replication origins are cis-acting elements that potentiate TNR instability; therefore, we mapped replication initiation sites and prereplication complex protein binding within the ~10-kb DMPK/SIX5 locus in non-DM1 and DM1 cells. Two origins, IS(DMPK) and IS(SIX5), flanked the (CTG)(n) · (CAG)(n) TNRs in control cells and in DM1 cells. Orc2 and Mcm4 bound near each of the replication initiation sites, but a dramatic change in (CTG)(n) · (CAG)(n) replication polarity was not correlated with TNR expansion. To test whether (CTG)(n) · (CAG)(n) TNRs are cis-acting elements of instability in human cells, model cell lines were created by integration of cassettes containing the c-myc replication origin and (CTG)(n) · (CAG)(n) TNRs in HeLa cells. Replication forks were slowed by (CTG)(n) · (CAG)(n) TNRs in a length-dependent manner independent of replication polarity, implying that expanded (CTG)(n) · (CAG)(n) TNRs lead to replication stress. Consistent with this prediction, TNR instability increased in the HeLa model cells and DM1 cells upon small interfering RNA (siRNA) knockdown of the fork stabilization protein Claspin, Timeless, or Tipin. These results suggest that aberrant DNA replication and TNR instability are linked in DM1 cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA. guoqi.liu@yahoo.com
authors.forename: Guoqi
authors.initials: G
authors.lastname: Liu
authors.forename: Xiaomi
authors.initials: X
authors.lastname: Chen
authors.forename: Yanzhe
authors.initials: Y
authors.lastname: Gao
authors.forename: Todd
authors.initials: T
authors.lastname: Lewis
authors.forename: Joanna
authors.initials: J
authors.lastname: Barthelemy
authors.forename: Michael
authors.initials: M
authors.lastname: Leffak
mesh_terms: D002460:Cell Line; D043171:Chromosomal Instability; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D018911:Trinucleotide Repeats
chemical_list: C585517:DMPK protein, human; D004247:DNA; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases