T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

Abstract

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase a. Several antibodies were identified that coprecipitated T antigen and DNA polymerase a, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase a also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.The initiation of viral DNA replication is an important, temporally regulated process during lytic infection of animal cells by DNA tumor viruses (8,58). Although the mechanisms that mediate DNA synthesis in eucaryotic cells are not well understood, in vitro replication assays for tumor virus DNAs have provided a useful tool for studying these processes. Major advances have been made by in vitro analyses of the biochemical steps leading to replication of the linear, double-stranded genome of adenovirus (7,8,51). However, the protein-priming mechanism used during adenovirus DNA synthesis is unlikely to be a general mechanism for cellular replication.By contrast, the small, circular DNA molecules of simian virus 40 (SV40) are of particular interest because several features suggest that the initiation of SV40 replication mimics cellular chromosomal replication. Bidirectional replication initiates from a single origin on the SV40 DNA, and the chromatin associated with the viral genome is similar to that bound to cellular DNA (13). Also, with the exception of the virus-coded large tumor antigen (T antigen), SV40 replication relies entirely on the cellular DNA replication apparatus.The 82,000-dalton T-antigen phosphoprotein has been implicated in a number of biological functions important for both lytic infection of permissive monkey cells and neoplastic transformation of nonpermissive rodent cells. Some of these functions have been mapped to different portions of the T-antigen gene, suggesting that the gene product contains several polypeptide domains that can act with some degree of independence. Three distinct biochemical activities are carried out by T antigen: (i) specific binding to multiple sites at the SV40 origin (48, 54, 56), (ii) ATP hydrolysis (9,20,57), and (iii) association with a 53,000-dalton host protein (29). The DNA binding activity is directly responsible for repression of early viral RNA synthesis, as demonstrated by in vitro transcription reactions supplemented with purified T antigen (23,44).A number of in vivo studies suggest that both the orig...

Casein kinase II phosphorylates DNA‐polymerase‐α–DNA‐primase without affecting its basic enzymic properties

“…In this regard, it is interesting to note that the phosphorylation of large tumour antigen by the cdc2 protein kinase stimulates simian virus 40 DNA replication [37 381. Furthermore, large T antigen forms a physical complex with DNA polymerase a [39,401. Such a complex, or complexes, between DNA polymerase a and other not-yet-known accessory proteins might be responsible for the stimulatory and inhibitory effects that have been observed upon phosphorylation and dephosphorylation of less pure preparations of the polymerase -primase.…”

Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication.

“…The extent of enhancement depends on the experimental parameters of the replication assays, such as the amount of DNA transfected (33), the duration of replication (6), or the presence of competing origins (25). The 21-and 72-bp repeats are not required for SV40 DNA replication in vitro (25,33,36).…”

“…These upstream repeated sequences are not necessary for origin function (36), although they enhance the extent of replication by 5-to 10-fold (3,6,9,19,25,33). The extent of enhancement depends on the experimental parameters of the replication assays, such as the amount of DNA transfected (33), the duration of replication (6), or the presence of competing origins (25). The 21-and 72-bp repeats are not required for SV40 DNA replication in vitro (25,33,36).…”

“…The extent of enhancement depends on the experimental parameters of the replication assays, such as the amount of DNA transfected (33), the duration of replication (6), or the presence of competing origins (25). The 21-and 72-bp repeats are not required for SV40 DNA replication in vitro (25,33,36 The experiments described in the present study were initiated to test the effect of upstream sequences on DNA replication potential in vivo of the AT-region deletion mutations. To this end viral genomes were reconstructed which contained wild-type sequences with the exception of the deletions at the late boundary of the A+T-rich region.…”

Function of DNA Polymerase α in a Replication Fork and its Putative Roles in Genomic Stability and Eukaryotic Evolution

“…3). SV40 large T antigen can form a complex with pol-, and stimulate its activity (Smale & Tjian, 1986;Dornreiter et al, 1990;Collins & Kelly, 1991;Dornreiter et al, 1992). Although DNA polymerase has been shown to exist in the cell nucleus throughout the cell cycle (Nakamura et al, 1984), it might act only in the S phase.…”