DNA replication in vitro - Europe PMC

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Jan 15, 1988 - With this in mind, we have used the papovavirus simian virus 40 (SV40) as a model for eukaryotic DNA replica- tion. The SV40 genome exists ...
The EMBO Journal vol.7 no.4 pp. 1 211 - 1 21 8, 1 988

Cellular factors required for multiple stages of SV40 DNA replication in vitro

Micaela P.Fairman and Bruce Stillman Cold Spring Harbor Laboratory, PO Box 100, Cold Spring Harbor, NY 11724, USA

Communicated by J.D.Watson

Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells. Upon fractionation of this extract, two essential replication factors have been identified. One of these is a multi-subunit DNA binding protein containing polypeptides of 70 000, 34 000 and 11 000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB). The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin. These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage X replication, and may reflect similar events in the replication of cellular chromosomes. Key words: SV40/DNA replication/T antigen

Introduction The mechanism and regulation of DNA replication, and the proteins involved in these events, have been extensively studied in various prokaryotic systems. These studies have led to the definition of distinct stages of initiation and elongation. In contrast, the events involved in eukaryotic chromosome replication have not been so well defined, although studies on the replication of animal virus chromosomes have been vigorously pursued because of their relative simplicity. The identification and characterization of cellular proteins which are required to replicate virus chromosomes may then lead to an understanding of their roles in normal chromosome replication. With this in mind, we have used the papovavirus simian virus 40 (SV40) as a model for eukaryotic DNA replication. The SV40 genome exists as a duplex molecule of 5.2 kb which replicates in the host nucleus as a circular molecule whose nucleosome structure and histone content is indistinguishable from those of their host (for review see DePamphilis and Bradley, 1986). SV40 replication is absolutely dependent on the SV40 A gene, which encodes the large tumor antigen (TAg), while all other components must be of cellular origin. T antigen is a 92 kd multifunctional protein which is required for the control of transcription (Tegtmeyer et al., 1975; Myers et al., 1981; Hansen et al., 1981) and cellular transformation, as well as DNA replication (Rigby and Lane, 1983). Binding of TAg to a defined sequence at ori (site II) is essential for the initiation of DNA replication, while the binding to another site (site 1) stimulates the rate of replication 2-fold. Biochemical properties of this -

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molecule include a nucleotide binding activity (Clertant et al., 1984), ATPase activity (Tjian and Robbins, 1979; Clark et al., 1981), and a DNA helicase activity (Stahl et al., 1986), and interactions with several cellular proteins have been demonstrated (Lane and Crawford, 1979; McCormick and Harlow, 1980; Smale and Tjian, 1986; Mitchell et al., 1987). The development of cell free systems for the replication of plasmids containing the SV40 origin of replication (ori) has been described for both monkey (Li and Kelly, 1984) and human cell extracts (Li and Kelly, 1985; Stillman and Gluzman, 1985; Wobbe et al., 1985). These extracts reproduce many features of SV40 replication found in vivo. Replication starts at a unique site (ori) and proceeds in a semi-conservative fashion bidirectionally around the molecule and terminates when the two forks meet (Stillman et al., 1985; Li and Kelly, 1985; Li et al., 1986; Decker et al., 1986). Multiple rounds of replication have been detected (Li and Kelly, 1984, 1985; Stillman and Gluzman, 1985; Wobbe et al., 1985). Fractionation of cellular extracts that can support ori and T antigen-dependent SV40 replication has resulted in the characterization of several proteins that were presumed to be required for SV40 replication, and has led to a clearer definition of their function. Murakami et al. (1986) have shown that addition of purified DNA polymerase c -primase complex from a permissive cell line (HeLa) will allow an extract from a non-permissive cell line (mouse) to support SV40 DNA replication, thus defining a role for the polymerase a -primase complex in determining the species specificity of SV40 replication in vitro. Similarly the addition of purified topoisomerases I and II to depleted cellular extracts has defined a role for each of these activities. Either the type I or II enzyme can provide the swivel activity necessary for replication fork progression; however, topoisomerase II is uniquely required for the segregation of newly synthesized daughter molecules (Yang et al., 1987). As T antigen is the only virus encoded protein required for SV40 DNA replication, all the other essential factors must be of cellular origin and therefore be present in the cellular extracts. Fractionation of these extracts allows the identification of previously unknown cellular replication proteins which can then be studied and their functions defined. Initial separation of the cellular extract from human 293 cells by phosphocellulose chromatography yielded two fractions (I and II), which were inactive on their own, but when combined, supported efficient SV40 replication (Prelich et al., 1987). Fraction II contained both DNA polymerase and DNA primase activities, as well as topoisomerases I and II, while fraction I contained proteins of unknown function which were also essential for SV40 replication in vitro. Fraction I was subsequently separated into two fractions, designated A and B. A single factor that is required for SV40 replication has been purified from fraction B and identified as the proliferating cell nuclear antigen, PCNA (Prelich et 1211

M.P.Fairman and B.Stillman

293 cells was separated into two components by phosphocellulose column chromatography (I + II, Figure IA and B). As fraction I contains proteins that are essential for SV40 DNA replication but were of unknown function, this fraction was studied in more detail. This fraction was further divided by DEAE-cellulose column chromatography into two components A and B. The combination of fractions A and II allowed partial replication of the template DNA, whereas the inclusion of either fraction B or purified PCNA resulted in a greater level of synthesis and the formation of full length replication products (Prelich et al., 1987; Prelich and Stillman, 1988). Conversely, in the absence of A, replication was not observed. To further characterize the essential components present in fraction A, it was further divided into Al and A2 by ammonium sulphate precipitation. All the necessary replication factors required to reconstitute DNA replication in the presence of fractions II and B were contained in Al (Figure 1B). Fractionation in this manner resulted in a reduction of the protein content between A and A1 by 50 % with only a 5-10% loss in total replication activity. Further separation of the replication components pre-

al., 1987). Previous experiments (Fairman et al., 1987) have established that factors in A, in conjunction with T antigen, are required for the initial stages of SV40 DNA replication and therefore this fraction has been examined in detail. This paper describes the fractionation of A into two components, both of which are essential for SV40 replication in vitro. One of these components has been purified and consists of a complex of three polypeptides that binds tightly to DNA and, although essential for replication, is not required for the first stage. The other fraction, which may contain multiple factors, is also indispensable for replication, and is required for the formation of an initiation complex at the SV40 ori region. The identification of these cellular proteins has defined a number of separate stages of SV40 DNA replication.

Results Fractionation of A As previously described (Fairman et al., 1987; Prelich et al., 1987), the cellular extract (S100) derived from human

Fractionation of Cellular Replication Corm

A

293 Cells

Si00 Phosphocel lulose

B

LIi

I DEAE I

B

Ammonium Sulphate

SS DNA Cellulose Mono Q

LI

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pmol dAMP h-1

s100

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I

1.3

II

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I+II

53.4

A+II

16.3

B+II

A+B+II

1.6 72.3

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A2+B+II

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A1+A2+B+II

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SSI+B+II

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SSIII+B+II

4.4

SSI+SSIII+B+II

63.2

Phenyl Sepharose

A2

A

Component

SSII

Glycerol Gradient DEAE | PCNA

]

Glycerol Gradient

RFi R

Fig. 1. Fractionation of the 293 cell extract into multiple components. (A) Flow diagram of the fractionation scheme. Fractionation of the S100 extract into fractions I, II, A and B and the purification of PCNA from B were as described by Prelich et al. (1987). The fractionation of A into the two essential components SSI and RF-A is described in Materials and methods. Briefly, fraction A was divided into Al and A2 by ammonium sulphate precipitation. Al was then further fractionated by denatured DNA cellulose chromatography to give fractions SSI, SSI and SSIII. SSIII was further purified by DEAE-cellulose chromatography and preparative glycerol gradient velocity sedimentation to give replication factor A (RF-A). (B) Amount of DNA replication obtained with reconstituted replication components. Optimal amounts of the indicated fractions were combined with pSVOIO plasmid DNA and T antigen and reacted for 1 h at 37°C. The amount of DNA synthesis was determined by acid precipitation and liquid scintillation counting. The DNA synthesis is shown

1212

as

pmol of dAMP incorporated.

SV40 DNA replication in vitro

sent in this fraction was achieved by denatured DNA cellulose column chromatography. Three protein fractions were obtained (SSI, SSII and SSIII), two of which (SSI and SSIII) were essential for reconstitution of SV40 DNA replication with fractions B and II (Figure iB). Fraction SSII had no effect on replication in this system either singly or in combination with the other fractions (data not shown). The data presented in Figure 2A show a titration of SSI using optimal amounts of B and II in the presence or absence of SSIII, demonstrating that SSIII is essential for DNA replication. Similarly, Figure 2B shows a titration of increasing amounts of SSIII with optimal amounts of B and II in the presence or absence of SSI, demonstrating that this fraction is also essential. Since SDS -polyacrylamide gel electrophoresis of protein fractions from the denatured DNA cellulose column indicated that SSIII contained only a few protein species, the replication activity was further purified by DEAE -cellulose chromatography and glycerol gradient velocity sedimentation to yield a single replication component. Fractions from such a glycerol gradient were assayed for their ability to support DNA synthesis in the presence of T antigen and fractions SSI, B and II and were also examined by SDS -PAGE followed by silver staining. A single peak of activity sedimenting at 5-5.3S was observed, which corresponds exactly with the migration of three polypeptides with apparent mol. wts of 70 000, 34 000 and 11 000 daltons (henceforth referred to as 70K, 34K and 11K polypeptides; Figure 3). Other experiments show that these three polypeptides co-migrated with each other and the replication activity on a wide variety of chromatographic resins and separation techniques (i.e. phosphocellulose, DEAE, native DNA cellulose, mono S and mono Q) and appear to be tightly coupled. An SDS -polyacrylamide gel of the purified proteins, similar to that shown in Figure 3, but stained with

Coomassie brilliant blue, was scanned at 600 nm to estimate the stoichiometry of the three proteins. Integration of the area under each peak revealed that the 70K, 34K and 11K proteins were present at a ratio of 1.0:0.8:1.4, suggesting that the multi-subunit complex contains the three polypeptides in a 1:1:1 ratio. The complex of the three purified polypeptides has been designated replication factor A (RFA), following consultation with T.Kelly, whose laboratory has recently identified a similar series of polypeptides (personal communication). This complex did not show either DNA helicase or ATPase activities nor does it bind to the SV40 ori core region in a sequence specific manner (data not shown). Identification of RF-A as a stable polypeptide complex As stated above, the three polypeptides that constitute RFA could not be separated by chromatography on a wide variety of resins. The stability of the complex was also demonstrated by its resistance to denaturation by urea. In this experiment (Figure 4), samples of RF-A were preincubated in the presence or absence of 6 M urea (final con-

centration) before sedimentation in glycerol gradients (without urea). As shown in panels B (absence of urea) and D (presence of urea) the three polypeptides still co-migrated as a single species in the gradient, indicating that the complex could not be disrupted by this denaturing agent. To ensure that the conditions used were sufficient to disrupt other multi-subunit proteins, selected markers were run in parallel gradients. The multi-subunit protein catalase, when denatured under identical conditions and subjected to glycerol gradient sedimentation, separated into its component subunits, thereby shifting its position in the gradient following urea denaturation relative to its sedimentation position using native conditions (compare panels Figure 4A - C).

B.

A.

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