Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

Abstract

Nuclear protein factor type 1 (NPF-1) thatsimulates IMR-32 primase-associated DNA polymerase a, and a2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase a, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37C), DNase II (60 min at 370C), and heat treatment (2 min at 68C). The '2I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase a-primase complex (a, and a2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of a-amanitin (400 ,tg/ml). 8.0/1 mM EDTA/6 mM KCI/2 mM MgCl2/1 mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluoride/0.1% polyethylene glycol (PEG; 8 kDa) using a glass-Teflon homogenizer. The homogenate was centrifuged at 100,000 x g for 1 hr. The supernatant (1.5 ml) was applied to a DE 23 column (1.5 x 10 cm) equilibrated with 50 mM potassium phosphate, pH 7.6/1 mM MgCl2/1 mM 2-mercaptoethanol/0.1% PEG/ 0.1 mM phenylmethylsulfonyl fluoride (buffer C). After

DNA Replication Machinery: Functional Characterization of a Complex Containing DNA Polymerase α, DNA Polymerase δ, and Replication Factor C Suggests an Asymmetric DNA Polymerase Dimer

“…Pol R has been purified by several eukaryotic sources as a four-subunit enzyme with associated DNA primase activity [reviewed in Wang (1991)], and it has been shown to be absolutely required for initiation of DNA replication both in Vitro and in ViVo (Johnson et al, 1985;Pizzagalli et al, 1988;Challberg & Kelly, 1989;Stillman, 1989). Pol R is likely to be part of a larger multiprotein complex, since several laboratories have isolated pol R as a larger complex containing a number of enzymatic activities such as pol, exonuclease, and ATPase (Hu ¨bscher et al, 1982;Baril et al, 1983;Pritchard & DePamphilis, 1983;Hu ¨bscher & Ottiger, 1984;Takada et al, 1986;Vishwanatha et al, 1986;Ottiger et al, 1987;Biswas & Biswas, 1988). In addition, a 21S enzyme complex has been isolated from HeLa cells which contains pol R, a DNase, DNA ligase, DNA topoisomerase I, RNase H, and PCNA and which is active in the SV40 in Vitro DNA replication system (Malkas et al, 1990).…”

Purification and characterization of a yeast DNA polymerase .alpha. complex with associated primase, 5'.fwdarw.3' exonuclease, and DNA-dependent ATPase activities

“…These four subunits may likely be a part of a larger multiprotein complex that represents the replicating form of polymerase a. In fact, studies in several laboratories have indicated that DNA polymerase a could be isolated as a large complex accompanied by a number of enzymatic activities such as polymerase, primase, and exonuclease (Hubscher et al, 1982;Baril et al, 1983; Takada et al, 1986;; Biswas & Biswas, 1988). The multimeric form of t This work was supported by a grant from the U.S. Public Health Service.…”

Molecular cloning of a gene encoding an ARS binding factor from the yeast Saccharomyces cerevisiae.

“…Sequence-specific DNA binding factors are involved in the control of various cellular processes, such as control of gene expression and DNA replication (1)(2)(3)(4)(5)(6)(7)(8)(9). Eukaryotic DNA replication initiates from multiple origins.…”