Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity

Abstract

We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)1 appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is * Corresponding author. origin-binding domain (residues 147 to about 247) (2, 49-52, 61) has been the best characterized. The maximal p53binding and helicase substrate-binding regions map to residues 272 to 517 (46) and 131 to 517 (75), respectively, and the ATP-binding region maps to amino acids (aa) 418 to 528 (4, 5). The first N-terminal 121 amino acids have been implicated in transactivation (57, 79), and a small region (residues 105 to 114) is responsible for binding to the Rb protein (13).One activity of T antigen that is less well characterized is its ability to bind nonspecifically to double-stranded and single-stranded DNAs (7,38,56). The exact function of this activity is not yet clear, but it has been implicated in origin-specific DNA binding (51). It may also be involved in subsequent steps during viral DNA replication, in the induction of cellular DNA synthesis, or in the activation of rRNA genes. We have mapped nonspecific DNA binding to a region between aa residues 131 and 517 (75). Recently, we showed that at least 5 residues (mapping between positions 149 and 203) within the DNA binding domain were important in binding nonspecifically to doubleand single-stranded DNAs (51,52). All mutations in the domain which significantly reduced nonspecific binding also prevented originspecific binding (although the converse was not true). This suggested that T antigen first binds to virus DNA nonspecifically and then locates the origin sequences.Several years ago, Prives et al. (44) reported that a 56-kDa adeno-SV40 hybrid protein (containing T-antigen sequences from about residues 252 to 708) bound calf thymus DNA but could not bind specifically to the SV40 origin. A smaller, 45-kDa protein, with T-antigen sequences from about residues 336 to 708 was unable to bind to any DNA. These 5443 Vol. 66,No. 9

Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin

“…It is interesting that T-ag-bd 131-260 interacts very poorly with the non-origin-containing DNA control fragments. This observation is consistent with studies by Lin et al (42), who concluded that a second region of T-ag, extending between residues 269 and 522, defines a region that is important for nonspecific DNA binding. Since T-ag-bd 131-260 lacks this region, this molecule would be expected to interact poorly with non-origin-containing DNA.…”

“…The best-defined and most extensively studied domain of T-ag is the origin DNA-binding domain (T-ag-bd) (2,49,60,71,81), also termed the T-ag-obd (44). Currently, there is some uncertainty regarding the exact boundaries of the T-ag-bd; it has been mapped to amino acids 131 to 259 (2), 132 to 246 (49), and 147 to approximately 247 (42). Nevertheless, peptides from all three size classes are known to be sufficient for sequence-specific binding to the SV40 origin region (2,33,49,72).…”

Differences in the Reactivity of CD4+ T‐Cell Lines Generated against Free Versus Nucleosome‐Bound SV40 Large T Antigen

“…SV40 T‐ag purified from Sf‐9 cells infected with recombinant baculovirus has been described previously [21, 26, 27]. The physical characteristics of recombinant T‐ag produced by Sf‐9 cells are similar to those for T‐ag expressed in mammalian cell lines, including DNA and p53 binding.…”

E1 initiator DNA binding specificity is unmasked by selective inhibition of non-specific DNA binding

“…Papillomavirus DNA replication shows very distinctive similarities to SV40 replication, and the structural similarities between the DBDs in these two proteins indicate that T‐ag and E1 are highly related (Luo et al ., 1996; Enemark et al ., 2000). It has been recognized that DNA‐binding activities other than those accounted for by the DBD may exist in SV40 T‐ag, but the nature and function of those activities, as well as their precise location, have been difficult to determine (Lin et al ., 1992). However, previous footprinting experiments with T‐ag generally are consistent with the manner of DNA binding that we propose for E1 (SenGupta and Borowiec, 1994).…”