Functions of replication factor C and proliferating-cell nuclear antigen : Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T 4 ( simian virus 40 DNA replication / DNA binding / ATPase / evolution )

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Author	Tsurimoto, Toshiki Stillman, Bruce W.
Abstract	The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase 8 on a primed singlestranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNAdependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4. At aDNA replication fork, appropriate assembly ofaccessory proteins and DNA polymerase (pol) is required to form an active holoenzyme (1, 2). Little was known about either the mechanism of eukaryotic DNA replication or the accessory proteins that interact with eukaryotic pols; however, recent studies on simian virus 40 (SV40) DNA replication in vitro have provided insights into DNA replication in mammalian cells (3). Although DNA polymerase a (pol a) was long thought to be a primary replicative poi in eukaryotes, the involvement ofDNA polymerase 6 (pol 8) in DNA replication was suggested only recently by studies with the SV40 system (4, 5) and has been demonstrated by genetic studies in yeast (6, 7). These studies suggest that a dimeric pol complex containing pol a, pol 8, and accessory proteins is responsible for coordinated replication of both the leading and lagging strands at the replication fork (3, 8). Pol 6 contains a 3'-5' exonuclease activity and can synthesize DNA processively on poly(dA)-oligo(dT) primer-template DNA in the presence of one accessory protein, proliferating-cell nuclear antigen (PCNA) (9, 10). Further studies demonstrated that pol 6 can synthesize DNA processively and efficiently on a primed M13 single-stranded DNA (ssDNA) only in the presence of three replication factors, replication factor A (RF-A), PCNA, and replication factor C (RF-C) (8). RF-A is a multisubunit ssDNA binding protein that is required for both initiation and elongation ofDNA replication in vitro and functions as a stimulatory factor for pol a and pol 6 (8, 11-15). This latter function may be analogous to the activities of a number of viral, phage, and bacterial ssDNA binding proteins that stimulate their homologous pols (2, 3, 16). RF-C is required, along with PCNA, only for the elongation stage ofSV40 DNA replication and appears to be required for the coordinated synthesis of leading and lagging strands (5, 17). Several properties of RF-C, for example, its function as a pol accessory protein and its moderate affinity for ssDNA bound to cellulose (17), suggested to us that it may be involved in template-primer recognition or as a molecular clamp holding the pol onto the template DNA. Analogous functions have been suggested for prokaryotic pol accessory proteins (1, 2, 16). Studies on Escherichia coli and its phages have elucidated the general mechanisms for the initiation and elongation of DNA replication. Particularly relevant to this discussion are the studies on the bacteriophage T4 DNA replication proteins required at the replication fork [for review, see Cha and Alberts (18)]. The phage pol, encoded by gene 43, synthesizes the leading and lagging strands at a replication fork, forming a dimeric pol complex. Pol accessory proteins, encoded by genes 44 and 62, function as a DNA-dependent ATPase and primer-recognition protein complex. This DNA-dependent ATPase activity is stimulated by another protein, encoded by gene 45, and together the gene 44/62 and gene 45 proteins complex and cooperate to stimulate the processivity of pol, forming a pol holoenzyme. In addition to these proteins, the helix-destabilizing protein (gene 32, ssDNA binding protein) and the "primosome" proteins, encoded by genes 41 and 61 (DNA helicase and primase), also function at the replication fork to unwind the parental duplex DNA, form primers for Okazaki fragment synthesis on the lagging strand, and augment pol function. We have further characterized two human cell replication factors, RF-C and PCNA, and demonstrate that they are strikingly similar to proteins encoded by the phage T4 genes 44/62 and 45, respectively. MATERIALS AND METHODS Replication Factors, RF-A, PCNA, and RF-C, and Pol 6. RF-A was purified from a human 293 cell cytoplasmic extract as described (15) and a ssDNA-cellulose fraction (650 tug/ml) was used. Two sources of PCNA were used in this study. PCNA (200 pug/ml) purified from human 293 cells by a published procedure (4) was used in Figs. 2 and 4. PCNA that had the same specific activity as PCNA from human cells was produced in E. coli harboring a plasmid carrying the human PCNA cDNA sequence (19) under the control of bacteriophage T7 promoter. The E. coli-produced PCNA was purified Abbreviations: pol, DNA polymerase; pol a, DNA polymerase a; pol 8, DNA polymerase 8; pol III, DNA polymerase Ill; RF-A, replication factor A; RF-C, replication factor C; PCNA, proliferating-cell nuclear antigen; SV40, simian virus 40; ATP[S], adenosine 5'[y-thio]triphosphate; ssDNA, single-stranded DNA. 1023 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1024 Biochemistry: Tsurimoto and Stillman to homogeneity by four steps (K. Fien and B.S., unpublished results) and used in Fig. 3. RF-C was purified from human 293 cell nuclear extracts through four steps (ssDNA-cellulose fraction; 60 or 40 ,ug of RF-C per ml) or five steps (glycerol gradient fraction; 8 ,ug of RF-C per ml) as published (17). Pol 8 was purified from calf thymus (90 g) by five steps as described (8, 20) and the ssDNA-cellulose fraction with a specific activity of 6.8 x 103 units/mg was used. One unit of pol activity was defined as the incorporation of 1 nmol of dTMP at 370C in 1 hr under conditions described in ref. 8. DNA Synthesis Reaction on a Primed M13 ssDNA with Pol 6. The reaction mixture (25 ,ul) containing 30 mM Hepes (pH 7.8), 30 mM NaCI, 7 mM MgCl2, 0.5 mM dithiothreitol, bovine serum albumin (0.1 mg/ml), all four dNTPs (each at 0.05 mM) with [a-32P]dATP (2000 cpm/pmol), 100 ng of M13 ssDNA (M13mpl8) primed with a 3-fold molar excess of a unique 17-base sequencing primer (primer 1211 from New England Biolabs), 200 ng of PCNA, 1 ug of RF-A and 0.27-0.54 unit of pol 8 was incubated at 37°C for 30 min, and acid-insoluble radioactivity was determined. ATPase Assay. The assay for RF-C ATPase was essentially the same as described (21). The reaction mixture (25 ul) containing 50 mM of Tris HCI (pH 7.9), 0.1 M NaCI, 1 mM dithiothreitol, 2 mM MgCl2, bovine serum albumin (100 ,ug/ml), 0.1 mM [_y-32P]ATP, and the indicated DNAs was incubated at 37°C for 30 min.
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