Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase δ Expressed in Baculovirus-infected Insect Cells

Abstract

The catalytic subunit of human DNA polymerase ␦ has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a M r ‫؍‬ ϳ125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase ␦. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3 -5 exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase ␦ isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn 2؉ or Mg 2؉ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn 2؉ than in Mg 2؉ . The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase ␦ are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase ␦ is essential for functional interaction with PCNA.Although genetic studies in yeast have suggested that three distinct DNA polymerases (␣, ␦, and ⑀) are required for nuclear DNA replication in eukaryotes (1-4), only two DNA polymerases (␣ and ␦) were found to be essential components of an in vitro reconstituted SV40 DNA replication system (5); pol 1 ␣, with its tightly associated primase activity, was shown to be required for the synthesis of primers for both leading and lagging strands, whereas pol ␦ and its accessory proteins, proliferating cell nuclear antigen (PCNA) and replication factor C, also known as activator 1, were found to be required for leading strand synthesis and for completing Okazaki fragments initiated by pol ␣/primase. More recently, elucidation of the roles of DNA polymerases ␣, ␦, and ⑀ in DNA replication was approached by UV cross-linking of polymerases to nascent DNA within replicating chromosomes (6). These studies showed that only pol ␣ and pol ␦ were photolabeled by nascent SV40 DNA whereas pol ␣, pol ␦, and pol ⑀ were all photolabeled by nascent cellular DNA, suggesting that the replication of SV40 chromosomes may require only a subset of the proteins required for replication of cellular chromosomes. Thus, the roles of pol ␦ and pol ⑀ in cellular DNA replication may not be resolved by studies of the replication of DNA viruses; rather, studies of the properties of the replicative DNA polymerases and their interactions with other replication proteins will be necessary to answer these questions.pol ␦ has been purified from a number of eukaryotic sources including calf thymus (7), budding yeast (8), mouse cells (9), and Drosophila melanogaster (10). The enzyme is usually isol...

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

“…Three of the four human pol δ subunits can interact directly with PCNA. The p66 accessory subunit contains a canonical PIP box, whereas the p125 catalytic subunit and the p12 accessory subunit both contain noncanonical PIP domains where the conserved Q residue has been replaced with an alternative amino acid (13)(14)(15)(16)(17)(18)(19)(20)(21). The homotrimeric PCNA sliding clamp contains three identical binding sites for PIP box-containing proteins and, hence, human pol δ may simultaneously bind all three subunits within a given PCNA trimer, similar to that observed in S. cerevisae (22).…”

“…The experiment for pol δ alone was performed identically except for the omission of PCNA and RFC. (B) 16% denaturing sequencing gel of the primer extension products for pol δ alone (lanes 1-6) and pol δ holoenzymes assembled with either PCNA (lanes 7-12) or (Ub) 3 -PCNA (lanes [13][14][15][16][17][18]. The sizes of the substrate and full-length product are indicated on the left and the insertion step (i) for each primer extension product up to 36 nt in length (i = 7) is indicated on the right.…”

Identification of a Fourth Subunit of Mammalian DNA Polymerase δ

“…prokaryotic or lower eukaryotes such as yeast. Recently, expression systems for the p125 (15,24,25), the p50 subunit (16), and the recombinant heterodimer have been developed (26). The p50 subunit has no known enzymatic functions, but has been shown to be required for the response of the p125 subunit to PCNA (16,26).…”

Ten1p promotes the telomeric DNA-binding activity of Cdc13p: implication for its function in telomere length regulation

“…The preparation of His-Ten1p is as described in "Purification of recombinant Ten1 in E. coli". The 585-bp fragment of the coding region of STN1 gene (900-1 485 nt) was PCR amplified with primers containing BamHI and XhoI restriction sites, and inserted into pGEX-4T-1 plasmid to produce GST-Stn1(300-495) p. The 726-bp fragment of the coding region of CDC13 (1-726 nt) was PCR amplified with primers containing BamHI restriction site, and inserted into pGEX-4T-1 plasmid to produce GSTCdc13(1-234)p. The purification of the antigens including HisTen1p, GST-Stn1(300-495)p and GST-Cdc13(1-234)p, the raise of antiserum and the affinity purification of specific antibodies with antigen column were as described previously [32,33].…”