Altered Replication in Human Cells Promotes DMPK (CTG)(n)center dot(CAG)(n) Repeat Instability

作者：Liu Guoqi; Chen Xiaomi; Gao Yanzhe; Lewis Todd; Barthelemy Joanna; Leffak Michael^*

来源：Molecular and Cellular Biology, 2012, 32(9): 1618-1632.

DOI：10.1128/MCB.06727-11

Myotonic dystrophy type 1 (DM1) is associated with expansion of (CTG)(n)center dot(CAG)(n) trinucleotide repeats (TNRs) in the 3%26apos; 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 similar to 10-kb DMPK/SIX5 locus in non-DM1 and DM1 cells. Two origins, ISDMPK and ISSIX5, flanked the (CTG)(n)center dot(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)center dot(CAG)(n) replication polarity was not correlated with TNR expansion. To test whether (CTG)(n)center dot(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)center dot(CAG)(n) TNRs in HeLa cells. Replication forks were slowed by (CTG)(n)center dot(CAG)(n) TNRs in a length-dependent manner independent of replication polarity, implying that expanded (CTG)(n)center dot(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.

• 出版日期2012-5