DNMT3b protects centromere integrity by restricting R-loop-mediated DNA damage.

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• DNA methyltransferase 3b (DNMT3b) plays a crucial role in maintaining genome stability.
• Dysfunction of DNMT3b is implicated in diseases like immunodeficiency-centromeric instability-facial anomalies syndrome (ICF).
• R-loops are implicated in genome instability, but their precise role in DNMT3b dysfunction is unclear.

Purpose of the Study:

• To investigate the mechanisms by which DNMT3b dysfunction leads to genome instability.
• To determine the role of R-loops in DNA damage in DNMT3b knockout and ICF cells.
• To elucidate the repair pathways of DNA double-strand breaks in centromeric regions in ICF cells.

Main Methods:

• Utilized DNMT3b knockout cells and ICF cells with DNMT3b mutations.
• Employed genome-wide ChIP-sequencing to map DNA damage sites.
• Analyzed R-loop levels and endonuclease activity.
• Investigated DNA double-strand break repair pathways.

Main Results:

• DNMT3b dysfunction and loss of expression, coupled with p53 mutation, increase DNA damage.
• DNA damage sites were mapped to satellite repetitive DNA sequences, particularly centromeric regions.
• Reduced steady-state levels of centromeric R-loops were observed, suggesting their removal.
• XPG and XPF endonucleases cleave R-loops, causing DNA breaks and chromosome instability.
• DNMT3b dysfunction increases susceptibility of R-loops to cleavage.
• Centromeric DNA double-strand breaks in ICF cells are likely repaired by error-prone end-joining.

Conclusions:

• DNMT3b dysfunction compromises centromere integrity through R-loop-mediated DNA damage.
• The cleavage of R-loops by XPG/XPF contributes to chromosome instability.
• Error-prone repair of centromeric breaks exacerbates genomic instability in ICF cells.