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

The change of repair efficiency of UV-induced pyrimidine dimers due to aging was examined in replicatively senesced fibroblasts. The fibroblasts with repeated passages showed the characteristics of cellular senescence, including irreversible cell cycle arrest, elevated β-galactosidase activity, and senescence-associated secretory phenotype. The incision efficiency of oligonucleotide containing UV lesions was similar regardless of cell doubling levels, but the gap filling process was impaired in replicatively senescent cells. The releases of xeroderma pigmentosum group G, proliferating cell nuclear antigen, and replication protein A from damaged sites were delayed, which might have disturbed the DNA polymerase progression. The persistent single-stranded DNA was likely converted to double-strand breaks, leading to ataxia telangiectasia-mutated phosphorylation and 53BP1 foci formation. Phosphorylated histone H2AX (γ-H2AX) induction mainly occurred in G1 phase in senescent cells, not in S phase such as in normal cells, indicating that replication stress-independent double-strand breaks might be formed. MRE11 having nuclease activity accumulated to damaged sites at early time point after UV irradiation but not released in senescent cells. The pharmacological studies using specific inhibitors for the nuclease activity suggested that MRE11 contributed to the enlargement of single-stranded DNA gap, facilitating the double-strand break formation.