Altered replication in human cells promotes DMPK (CTG)(n) · (CAG)(n) repeat instability.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Myotonic dystrophy type 1 (DM1) is characterized by expanded trinucleotide repeats (TNRs) in the DMPK gene.
• Replication origins influence TNR instability, prompting investigation into their role in DM1.

Purpose of the Study:

• To map replication initiation sites and protein binding within the DMPK/SIX5 locus in DM1 and control cells.
• To determine if TNRs act as cis-acting elements of instability in human cells.
• To investigate the link between DNA replication stress and TNR instability in DM1.

Main Methods:

• Mapping replication origins and protein binding (Orc2, Mcm4) in DM1 and non-DM1 cells.
• Creating HeLa cell models with integrated (CTG)(n) · (CAG)(n) TNRs and a c-myc replication origin.
• Assessing replication fork speed and TNR instability after siRNA knockdown of fork stabilization proteins (Claspin, Timeless, Tipin).

Main Results:

• Two replication origins, IS(DMPK) and IS(SIX5), were identified flanking the TNRs in both cell types.
• Expanded TNRs slowed replication forks in a length-dependent manner, independent of replication polarity.
• TNR instability increased upon knockdown of fork stabilization proteins in model and DM1 cells.

Conclusions:

• Expanded (CTG)(n) · (CAG)(n) TNRs induce replication stress.
• Aberrant DNA replication and TNR instability are interconnected in DM1 pathogenesis.
• Replication fork stabilization pathways are crucial for maintaining TNR stability.