DNA ligase I deficiency leads to replication-dependent DNA damage and impacts cell morphology without blocking cell cycle progression.
46BR.1G1 cells derive from a patient with a genetic syndrome characterized by drastically reduced replicative DNA ligase I (LigI) activity and delayed joining of Okazaki fragments. Here we show that the replication defect in 46BR.1G1 cells results in the accumulation of both single-stranded and double-stranded DNA breaks. This is accompanied by phosphorylation of the H2AX histone variant and the formation of gammaH2AX foci that mark damaged DNA. Single-cell analysis demonstrates that the number of gammaH2AX foci in LigI-defective cells fluctuates during the cell cycle: they form in S phase, persist in mitosis, and eventually diminish in G(1) phase. Notably, replication-dependent DNA damage in 46BR.1G1 cells only moderately delays cell cycle progression and does not activate the S-phase-specific ATR/Chk1 checkpoint pathway that also monitors the execution of mitosis. In contrast, the ATM/Chk2 pathway is activated. The phenotype of 46BR.1G1 cells is efficiently corrected by the wild-type LigI but is worsened by a LigI mutant that mimics the hyperphosphorylated enzyme in M phase. Notably, the expression of the phosphomimetic mutant drastically affects cell morphology and the organization of the cytoskeleton, unveiling an unexpected link between endogenous DNA damage and the structural organization of the cell.
Soza
Samuela
S
Istituto di Genetica Molecolare, Consiglio Nazionale della Ricerche, Pavia, Italy.
Leva
Valentina
V
Vago
Riccardo
R
Ferrari
Giovanni
G
Mazzini
Giuliano
G
Biamonti
Giuseppe
G
Montecucco
Alessandra
A
D002453:Cell Cycle; D002460:Cell Line; D048430:Cell Shape; D003599:Cytoskeleton / Q000473:pathology; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000172:deficiency*; D004261:DNA Replication; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D050505:Mutant Proteins; D010766:Phosphorylation; D012867:Skin / Q000166:cytology
C468783:H2AX protein, human; D006657:Histones; C000607892:LIG1 protein, human; D050505:Mutant Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP