Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py)...

Area of Science:

• Genetics
• Molecular Biology
• Genomic Instability

Background:

• DNA sequences forming noncanonical structures, such as hairpins and G-quadruplexes, are linked to replication fork stalling, DNA damage responses, and genomic instability in human diseases.
• The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat in the human polycystic kidney disease (PKD1) intron 21 is known to form non-B DNA secondary structures in vitro.

Purpose of the Study:

• To investigate the in vitro and in vivo effects of the PKD1 mirror repeat on DNA replication fork dynamics.
• To determine if the PKD1 mirror repeat acts as a replication fork barrier and triggers DNA damage responses.
• To examine the consequences of replication fork stalling and subsequent checkpoint activation on cellular adaptation and mutation accumulation.

Main Methods:

• Integration of the PKD1 Pu-Py mirror repeat tract into the HeLa genome alongside the c-myc replicator at an ectopic chromosomal site.
• Analysis of replication fork stalling using in vitro and in vivo replication assays.
• Assessment of DNA damage response activation through the detection of replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) protein binding.
• Monitoring of checkpoint activation (Chk1 phosphorylation) and cellular adaptation.
• Evaluation of the effect of mirror repeat excision on DNA damage response.

Main Results:

• The PKD1 mirror repeat tract functions as a polar replication fork barrier in an orientation-dependent manner.
• Replication fork stalling at the mirror repeat activates the DNA damage response, evidenced by increased binding of RPA, Rad9, and ATR.
• Checkpoint activation (Chk1 phosphorylation) occurs independently of the proximal c-myc origin of replication.
• Cells with the replication fork barrier exhibit checkpoint adaptation, characterized by constitutive Chk1 phosphorylation and continued growth.
• Excision of the Pu-Py mirror repeat abrogates the DNA damage response.

Conclusions:

• The PKD1 mirror repeat is a functional replication fork barrier that induces DNA damage responses and checkpoint adaptation.
• Checkpoint adaptation in response to the PKD1 mirror repeat may permit the accumulation of mutations, contributing to genomic instability and disease.
• Understanding these mechanisms is crucial for comprehending the role of repetitive DNA elements in human genetic disorders.