Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations at the WRN gene. Several recent reports suggest that accumulation of DNA damage could lead to premature cellular aging. Therefore, WRN might function in DNA damage response, particularly DNA repair. Here, we investigated the role of WRN in DNA repair and genome integrity. WRN protein rapidly accumulated at DNA damage sites and formed discrete nuclear foci only during S phase, but not in G1 phase. WRN-defective WS cells showed the spontaneous accumulation of -H2AX (DSB marker), suggesting that WRN could function to repair the S phase-dependent DNA damage. However, WS cells showed homologous recombination (HR) at normal level, although HR repair functions preferentially during the S phase. Translesion DNA synthesis (TLS) is known as another repair pathway for S phase-dependent DNA damage. WS cells exhibit an increase in spontaneous focus formation of pol and Rad18, which are important for TLS regulation. WS cells also showed the spontaneous ubiquitination of PCNA and increased pol-related gene mutation. Taken together, WRN could work for the regulation of TLS pathway and might also be important to maintain genome integrity under a little DNA damage by tritium.