Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen

Abstract

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.The tumor antigen (T-antigen) of simian virus 40 (SV40), encoded by the viralA gene (1,18,23,24,32), binds to SV40 DNA (3) and is required for the initiation of viral DNA synthesis (4, 31). DNA replication is initiated at a unique site located 0.67 fractional unit from the EcoRI site on the conventional physical map of SV40 (11). Curiously, however, T-antigen binds with high affinity to three different HindII + HI restriction fragments of SV40 DNA, only one of which includes the origin of DNA replication (16). We have therefore examined the interaction of Tantigen with SV40 chromatin to determine whether the condensation of DNA into chromatin affected its capacity to bind T-antigen.It has been demonstrated that the histones of SV40 chromatin isolated from infected cells (9, 12) or from visions (5) are condensed into 21 spherical units or v-bodies. It is presumably these structures that result in the failure of staphylococcal nuclease to digest completely all ofthe DNA ofchromatin. Polisky and McCarthy (26) have argued that the protected regions are randomly distributed along the DNA; however, the chromatin they studied was prepared from virions by a technique that disrupts the v-bodies (5). We have therefore reexamined the distribution of the protected regions preparatory to the study of T-antigen binding to chromatin. MATERIALS AND ME]HODSCell growth. BSC-1 monkey kidney cells were grown in 75-cm2 tissue culture flasks (Falcon Plastics, Oxnard, Calif.) or 700-cm2 glass roller bottles in Eagle minimal essential medium (MEM; NIH Media Supply) supplemented with 2% (MEM-2) or 10% (MEM-10) fetal bovine serum (Flow Laboratories, Inc., Rockville, Md.) in ...

Structural changes in simian virus 40 chromatin as probed by restriction endonucleases

“…Furthermore, chromatin with such a distribution of histones should yield equimolar amounts of each limit digest DNA fragment after cleavage by multiple-site restriction enzymes. It should be pointed out that others (11,(41)(42)(43)(44) have presented similar arguments in previous studies on the arrangement of histones in SV40 and polyoma chromatin.…”

“…The additional 25% which is cleaved upon prolonged incubation suggests that regions presumably near, or perhaps within the nucleosome, are less accessible for cleavage by the enzyme. Importantly, the total susceptible fraction of 50% corresponds well with the estimate of coverage as determined by digestion with nonrestricted endonucleases (8,9,32,41).…”

“…They suggested that the cleavage was occurring only in the internucleosomal DNA regions. Nevertheless, Persico-Dilauro et al (41) found that 50% of DNA within intracellular SV40 chromatin was cleaved by both staphylococcal nuclease and EcoRI nuclease. From their analysis of the HindlIl digest patterns, they proposed that the nucleosomes are distributed randomly in the population of chromatin, but are not randomly arranged within the SV40 chromatin molecule.…”

Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants

“…Restriction digest analysis of viral DNA. Viral DNA samples were isolated and digested with restriction endonucleases as previously described (13,24). DNA samples of 10 ul contained 1 mg of bromophenol blue per ml, 12.5% glycerol, 2.5% sodium dodecyl sulfate, 50 mM EDTA, and 10 mM Tris-hydrochloride at pH 8.9.…”

Sequential stimulation of cellular RNA synthesis in polyoma-infected mouse kidney cell cultures

“…Microinjection of the transcripts from the early strand of SV40 DNA into quiescent mouse kidney cells led to synthesis of SV40 T-antigen, followed by stimulation of cellular DNA synthesis, indicating a causative relationship (19). It was shown that large SV40 T-antigen initiates SV40 DNA replication (20) and negatively controls early SV40 mRNA synthesis (21) and that both events involve the cooperative binding of large SV40 T-antigen to the origin of replication of the viral DNA (22,23). It was reported that large SV40 T-antigen has ATPase activity (24,25), that it interacts with a 53'000 dalton cellular protein (26,27), that it exerts a positive and direct control on the transcription of mammalian ribosomal genes (8,28) and that it positively controls the synthesis of several cellular polyA RNAs (29).…”