The DNA damage response (DDR) is a multifaceted network of signals which is activated by structural and chemical alterations of DNA. The DDR involves DNA repair, DNA damage tolerance and checkpoint pathways which act in coordination to overcome genotoxic stress. It is unclear if all alternative DNA polymerases (Alt. Pols) have complete overlapped functions in DDR. By using siRNA technology we depleted the expression of 6 different Alt. Pols, either individually or in combination, and evaluated their relevant contribution to DDR after the exposure to DNA damaging agents. We analysed the induction of known replication stress markers such as the phosphorylation of H2AX (γH2AX) and the recruitment of 53BP1 to foci. From all of the alt. Pols evaluated, only Alt. DNA polymerase iota (Pol ι) contributed differently to the induction of replication stress markers, were we observed reduced amount of γH2AX and less recruitment of 53BP1 to foci in the absence of Pol ι. We demonstrated that Pol ι prevents unleashed fork elongation, an unexpected result, as lack of alt. DNA pols leads to reduced fork elongation which in turns promotes checkpoint activation and the accumulation of replication stress markers. In contrast lack of Pol iota leads to less checkpoint activation, increased cell death and genomic instability. All together our data suggests that Pol iota is implicated in the coordination of checkpoint signals that arise from elongating forks to promote the correct onset of DDR.