Isolation and identification of the third subunit of mammalian DNA polymerase   by PCNA-affinity chromatography of mouse FM3A cell extracts

Abstract

Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

A tumor necrosis factor α- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase δ and proliferating cell nuclear antigen

“…However, PDIP1 shares only 20% identity with the mouse homolog of the pol ␦ third subunit, p66 (14), and does not appear to be an integral component of pol ␦ because it does not copurify with the enzyme from calf thymus (unpublished observation). Thus, PDIP1 is unlikely to be a homolog of Cdc27.…”

“…It is believed that, in addition to their roles in mediating the interaction of PCNA and pol ␦, both Cdc27 and Pol32 function as dimerization factors for pol ␦, a requirement for concurrent replication of the leading and lagging strands at the replication fork (12,13). A putative third subunit of mammalian pol ␦, p66, has been isolated from mouse (14) and calf thymus tissues (15) by affinity chromatography, but it is not yet clear whether mammalian p66 is functionally analogous to Cdc27 and Pol32.…”

“…[␣-32 P]dCTP and [ 3 H]dTTP were from ICN. Poly(dA) Ϸ2500 and oligo(dT) [12][13][14][15][16][17][18] were from Midland Certified Reagents (Midland, TX).…”

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

“…Mammalian Pol d was subsequently shown to be a four-subunit enzyme, Pol d4, by the identification of p68, a homolog of Pol32 [Hughes et al, 1999;Mo et al, 2000], and p12, which possesses limited sequence similarity to Cdm1 . Human Pol d4 is a heterotetramer of p125, p50, p68, and p12 subunits, like yPold4 in S. pombe.…”

FGF2-induced upregulation of DNA polymerase-δ p12 subunit in endothelial cells

“…Also, p125 subunit interacts with and becomes phosphorylated by cyclin D3/Cdk4 and cyclin E/Cdk2 (Wu et al, 1998), thus suggesting a possible role in S phase checkpoint control. Additionally, p66/68 (Hughes et al, 1999) and p12 were identified as part of the mammalian pold complex. Whereas p66/68 binds PCNA, a specific role for p12 subunit has not been identified, although its addition to an in vitro assay enhances the DNA polymerizing activity of the enzyme (Podust et al, 2002).…”