Our cells endure continuous challenges from byproducts of endogenous metabolic processes as well as outside sources such as UV light and environmental polltants. The DNA Damage Response (DDR) helps organisms cope with damage and coordinate an appropriate response pathway. It involves the initial detection of DNA damage and subsequent activation of the appropriate repair pathway, coordination of cell cycle progression, transcription activation, or activation of apoptosis or cellular senescence. ATR-Chk1 is one of the most studied DDR pathways and controls progression through the S-phase of the cell cycle. The translesion synthesis (TLS) pathway, once believed to act independently of the DDR pathways, ensures bypass of a DNA lesion in a rapid and often error-prone fashion. However, it remains largely unknown the interplay between the DDR and TLS pathways. In this research, we investigated the roles of two TLS polymerases, REV1 and Polη, to determine their function in the ATR-Chk1 pathway. Our data suggest that REV1 is important for the activation of ATR-Chk1 DDR pathway in response to interstand crosslinks and stalled replication forks, but is dispensable for the recruitment of checkpoint protein complex onto DNA damage sites or stalled forks. In addition, Polη is important for Chk1 phosphorylation in response to oxidative stress but not DNA replication stress. This research will help to understand how the TLS polymerases contribute to the DDR pathway to maintain genomic stability. Thus, our findings from this study will provide insight into how cells respond to chemotherapy drugs and environmental agents, and ultimately lead to new potential drug targeting mechanisms.