Poldip2: an investigation of structure and signaling pathways to elucidate the function of a novel cell cycle regulator

Polymerase-delta interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication and ROS production via Nox4. My goal is to clarify Poldip2 function by analyzing protein structure and Poldip2 regulated signaling pathways.;An in silico analysis of Poldip2 structure uncovered a highly conserved region on the surface of the protein that resembles a binding pocket. This region is solvent accessible, opening the possibility of Poldip2 binding an ion, small molecule or having enzymatic activity.;A gene microarray analysis comparing wild type and Poldip2 knockout samples uncovered key pathways such as circadian rhythms, cell cycle, mitochondrial function and metabolism that are affected by Poldip2 depletion. A further analysis of Poldip2 function in the cell cycle was performed.;Data presented in this study support a novel role for Poldip2 in the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2-/- embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2-/- mouse embryonic fibroblasts exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to senescence, as measured by p16 and p19 expression. There was also no change in apoptosis by Annexin V staining. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2-/- cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Cdk1 and CyclinA2 are downregulated in Poldip2-/- cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21 expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative diseases such as cancer and atherosclerosis.