The small subunit is required for functional interaction of DNA polymerase δ with the proliferating cell nuclear antigen

Author： Zhou Jin-Qiu He Hua Tan Cheng-Keat Downey Kathleen M. So Antero G.

Publisher： Oxford University Press

ISSN： 1362-4962

Source： Nucleic Acids Research, Vol.25, Iss.6, 1997-01, pp. : 1094-1099

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Abstract

DNA polymerase δ is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase δ (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase δ, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase δ in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase δ results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase δ with PCNA and for highly processive DNA synthesis.