Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme.
Mammalian DNA polymerase delta was originally characterized as a tightly associated heterodimer consisting of the catalytic subunit, p125, and the p50 subunit. Recently, two additional subunits, the third (p68) and fourth subunits (p12), have been identified. The heterotetrameric human pol delta complex was reconstituted by overexpression of the four subunits in Sf9 cells, followed by purification to near-homogeneity using FPLC chromatography. The properties of the four-subunit enzyme were shown to be functionally indistinguishable from those of pol delta isolated from calf thymus. The physicochemical properties of both the reconstituted heterotetramer and the heterodimer of the p125 and p50 subunits were examined by gel filtration and glycerol gradient ultracentrifugation. These studies show quite clearly that the heterodimer and heterotetramer complexes do not behave in solution as dimeric structures. This issue is of significance because several studies of the yeast pol delta complexes have indicated that the third subunit is able to bring about the dimerization of the pol delta complex. The heterodimer is only weakly stimulated by PCNA, whereas the heterotetramer is strongly stimulated to a level with a specific activity comparable to that of the calf thymus enzyme. These results resolve earlier, conflicting reports on the response of the heterodimer to PCNA. Nevertheless, the heterodimer does have some ability to interact functionally with PCNA, consistent with evidence that the p125 subunit itself has an ability to interact with PCNA. The functional interaction of PCNA with the pol delta complex may likely involve multiple contacts.
Xie
Bin
B
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Mazloum
Nayef
N
Liu
Li
L
Rahmeh
Amal
A
Li
Hao
H
Lee
Marietta Y W T
MY
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D006367:HeLa Cells; D020035:Holoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D020035:Holoenzymes; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D011994:Recombinant Proteins; D004258:DNA Polymerase III