Faulty Gap Filling in Nucleotide Excision Repair Leads to Double-Strand Break Formation in Senescent Cells

Area of Science:

• Molecular Biology
• Cellular Aging
• DNA Repair Mechanisms

Background:

• Cellular senescence, characterized by irreversible cell cycle arrest and specific molecular markers, is associated with aging.
• DNA damage accumulation is a hallmark of aging, but the efficiency of DNA repair in senescent cells remains incompletely understood.

Purpose of the Study:

• To investigate the impact of replicative senescence on the repair efficiency of UV-induced DNA damage, specifically pyrimidine dimers.
• To elucidate the molecular mechanisms underlying impaired DNA repair in senescent fibroblasts.

Main Methods:

• Utilized replicatively senescent human fibroblasts exhibiting hallmarks of cellular senescence.
• Assessed DNA repair kinetics, including incision and gap-filling steps, following UV irradiation.
• Analyzed the recruitment and release dynamics of key DNA repair proteins (XPG, PCNA, RPA) at damaged sites.
• Investigated the formation of DNA double-strand breaks (DSBs) and associated signaling pathways (γ-H2AX, ATM, 53BP1).
• Examined the role of MRE11 nuclease activity in DNA repair and DSB formation using pharmacological inhibitors.

Main Results:

• Senescent fibroblasts showed normal incision of UV lesions but impaired gap-filling, indicating a defect in post-incision repair.
• Delayed release of XPG, PCNA, and RPA from UV-damaged sites suggested stalled DNA polymerase progression.
• Increased formation of γ-H2AX foci, particularly in the G1 phase, pointed towards replication stress-independent double-strand breaks.
• MRE11 accumulated at damaged sites and its nuclease activity contributed to the enlargement of single-stranded DNA gaps, promoting double-strand break formation.

Conclusions:

• Replicative senescence compromises the gap-filling step of nucleotide excision repair for UV-induced DNA damage.
• Impaired DNA repair in senescent cells may involve stalled DNA polymerases and aberrant MRE11 activity, leading to double-strand break formation.
• These findings highlight a potential link between defective DNA repair and the aging process, contributing to genomic instability in senescent cells.