Mammalian DNA polymerase delta (pol delta) is essential for DNA replication, though the functions of this smallest subunit of POLD4 have been elusive. We investigated pol delta activities in vitro and found that it was less active in the absence of POLD4, irrespective of the presence of the accessory protein PCNA. shRNA-mediated reduction of POLD4 resulted in a marked decrease in colony formation activity by Calu6, ACC-LC-319, and PC-10 cells. We also found that POLD4 reduction was associated with an increased population of karyomere-like cells, which may be an indication of DNA replication stress and/or DNA damage. The karyomere-like cells retained an ability to progress through the cell cycle, Suggesting that POLD4 reduction induces modest genomic instability, while allowing cells to grow until DNA damage reaches an intolerant level. Our results indicate that POLD4 is required for the in vitro pol delta activity. and that it functions in cell proliferation and maintenance of genomic stability of human cells. (C) 2009 Elsevier Inc. All rights reserved