Stable G-quadruplex DNA structures promote replication-dependent genome instability.

Area of Science:

• Genomics
• Molecular Biology
• DNA Structure and Stability

Background:

• G-quadruplex (G4) and triplex (H3) DNA structures are prevalent in mammalian genomes.
• These non-B DNA conformations are implicated in regulating DNA replication, transcription, and overall genome stability.
• Understanding their role is crucial for deciphering mechanisms of genomic instability.

Purpose of the Study:

• To investigate the impact of G4 and H3 structures on genomic instability using an ectopic repeat tract from the PKD1 locus.
• To determine the role of Mus81 nuclease in DNA double-strand breaks at these ectopic sites.
• To differentiate the instability mechanisms induced by H3 versus G4 structures under replication stress.

Main Methods:

• Construction of cell lines with ectopic homopurine/homopyrimidine repeat tracts capable of forming H3 and G4 structures.
• Ligand-mediated stabilization of G4 structures to observe effects on DNA sequence.
• Analysis of DNA double-strand breaks, hypermutation, and translocations using molecular techniques.
• Comparison of genomic instability in H3 and G4 cell lines under normal growth and replication stress.

Main Results:

• Ligand stabilization of G4 structures led to deletions of the G4 sequence and kilobase-scale deletions.
• DNA double-strand breaks at the ectopic site were dependent on the Mus81 nuclease.
• Hypermutation and microhomology-mediated translocations were observed at the G4 site.
• G4 cell lines rapidly lost the G4 consensus sequence, while H3 cells showed instability and delayed reporter gene loss.

Conclusions:

• Both H3 and G4 non-B DNA conformations contribute to genomic instability.
• These structures exhibit differential responses to endogenous replication stress.
• Replication-dependent double-strand breaks at non-B DNA sites model instability seen in microhomology-mediated break-induced replication (BIR).