BSTRACTIn human cells, appropriate monomethylation of histone H4 lysine 20 by PrSet7 (also known as SET8 and SETD7) is important for the correct transcription of specific genes and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with proliferating cell nuclear antigen (PCNA), which targets PrSet7 for destruction through the pathway mediated by CRL4Cdt2 (the cullin ring finger ligase-4 complex containing Cdt2).However, the factors involved in positive regulation of PrSet7 histone methylation remain undefined. Here, we present biochemical and genetic evidence for a previously undocumented interaction between Drosophila PrSet7 (dPrSet7) and DNA polymerase a in Drosophila. Depletion of the polymerase reduces H4K20 monomethylation suggesting that it is required for dPrSet7 histone methylation activity. We also show that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin-bound dPrSet7 protein as S phase progresses. Based on these data we suggest that interaction with the DNA polymerase represents an important route for stimulation of PrSet7 histone methylase activity that is mediated by allowing loading of dPrSet7 onto chromatin or its subsequent activation.