The second-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 alpha is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits 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 the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase alpha complex.
Mizuno
T
T
The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan.
Ito
N
N
Yokoi
M
M
Kobayashi
A
A
Tamai
K
K
Miyazawa
H
H
Hanaoka
F
F
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002467:Cell Nucleus / Q000201:enzymology*; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion; D014162:Transfection
D019915:DNA Primase; D004256:DNA Polymerase I