Abstract Natural killer cells are critical components of the innate immune system that kill virally infected or malignant cells. Though rare, natural killer cell deficiency (NKD) occurs when NK cell development and function are impaired and individuals with NKD are susceptible to severe and recurrent viral infections. Several gene deficiencies have been described to result in NKD, including variants in MCM4, GINS1, MCM10and GINS4, which are components of the CMG helicase. The helicase unwinds DNA during replication and is found in any actively proliferating cell. NK cells are more strongly impacted by mutational deficiencies in helicase proteins than other lymphocytes, though the mechanisms underlying this are not completely understood. NK cells from individuals with NKD as a result of helicase deficiency have increased DNA damage, cell cycle arrest, and replication stress. Here, we induced replication stress during NK or T cell specific activation by chemical methods, using aphidicolin, and genetic methods like siRNA targeting helicase proteins. We found that the CD56 brightsubset of NK cells accumulates more DNA damage and replication stress during activation than CD56 dimNK cells or activated T cells. The drug aphidicolin increases apoptosis of CD56 brightNK cells through caspase activation while simultaneously decreasing intracellular perforin expression, a protein important for killing of target cells. This effect is modeled by siRNA mediated knockdown of CMG proteins, thus linking decreased protein expression to replication stress and impaired NK cell function. These findings lend important insight into the mechanisms by which helicase deficiencies selectively affect human NK cell function and cause NK cell deficiency. NIH R01A137275