THE 2ND-LARGEST SUBUNIT OF THE MOUSE DNA-POLYMERASE ALPHA-PRIMASE COMPLEX FACILITATES BOTH PRODUCTION AND NUCLEAR TRANSLOCATION OF THE CATALYTIC SUBUNIT OF DNA-POLYMERASE-ALPHA

DNA polymerase alpha-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase a lpha is made up of four subunits, the largest of which is the catalyti c subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physi ological role has remained unclear up until now. We set out to charact erize these subunits individually or in combination by using a cDNA ex pression system in cultured mammalian cells, Coexpression of p68 marke dly increased the protein level of p180, with the result that ectopica lly generated DNA polymerase activity was dramatically increased. Immu nofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunit s resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into t he nucleus. These results indicate that association of p180 with p68 i s important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synth esized DNA polymerase alpha complex.