DNA binding properties and replication activity of the T antigen related D2 phosphoprotein

Abstract

According to earlier genetic experiments, a region within the N-terminal 50-100 amino acids may be important for the replication function of T antigen, the initiator protein of simian virus 40 (SV40). We have investigated this possibility using the T antigen related D2 protein in several biochemical assay systems. D2 protein, a phosphoprotein coded for by the adeno-SV40 hybrid virus Ad2+D2, shares its 594 C-terminal amino acids with authentic T antigen and its 104 N-terminal amino acids with an adenovirus structural protein. We confirmed earlier studies showing that D2 protein appeared to bind well to specific binding sites in the SV40 origin of replication. We found, however, that D2 protein was rather inefficient, inducing the unwinding of the double-stranded origin region, and was much less active than authentic T antigen as an initiator of in vitro SV40 DNA replication. We interpret these findings to indicate that D2 protein molecules associate with the origin to form an aberrant complex that is quite inefficient, inducing DNA unwinding and the establishment of replication forks. The possibility that the N-terminus may be required for an optimal arrangement of T antigen at the origin was supported by results of dephosphorylation studies. Dephosphorylation of N-terminal phosphoamino acids had significant effects on the stability of D2 protein-origin complexes.

Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen.

“…Biochemical studies have indicated that 80%o of the phosphate moieties on the molecule can be removed by treatment with calf intestinal alkaline phosphatase (CLAP), while the remaining 20%6 are refractory to enzymatic dephosphorylation (12)(13)(14)(15). Furthermore, analysis of CIAP-treated protein revealed an increase in its ability to direct origin-specific DNA synthesis in vitro (13,14).…”

“…Furthermore, analysis of CIAP-treated protein revealed an increase in its ability to direct origin-specific DNA synthesis in vitro (13,14). Although the ATPase activity of the CIAP-treated protein was not altered (13)(14)(15), its ability to bind specific DNA sequences in the SV40 origin of replication increased severalfold (13,15,16). The SV40 origin of replication contains two T antigen binding sites (17,18).…”

“…Site II also functions in transcriptional control but is primarily an essential cis element within the minimal SV40 origin of replication (20,22). CLAP-treated T antigen displays a 4-fold increase in binding to site II and a 2-fold increase in binding to site 1 (13,15).Phospho amino acid analysis performed in several laboratories has confirmed that the phosphates present on serine residues are removed by CIAP treatment, while those covalently attached to the two threonine residues (124 and 701) are unaffected (12,14,15). Furthermore, genetic studies have indicated that, while Thr-701 is dispensible for T antigen function and virus viability (23,24), Thr-124 is absolutely essential (24, 25).…”

Evidence for Nucleic Acid Binding Ability and Nucleosome Association of Bombyx mori Nucleopolyhedrovirus BRO Proteins

“…Therefore, their ubiquitination and involvement in proteasome-directed cleavage could protect other viral proteins from degradation and increase the efficacy of the infection. Phosphorylation of BRO proteins may also regulate DNA and RNA binding activity as shown in many DNA binding proteins as well as LEF-3 and DBP of baculovirus (5,14,15;Zemskov,unpublished). A switch of functions might be modulated by factors such as the ratio of host DNA to viral DNA, interaction with specific proteins, and posttranslational modifications (phosphorylation and ubiquitination).…”

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

“…The phosphorylated residues of Tag reside in two clusters, one each at the amino and carboxy termini of the protein (55), and both lie outside the DNA-binding domain. Phosphorylation of these residues affects the ability of Tag to bind origin sequences (34,44,63) and support replication of SV40 origin-containing DNA (24,44). Partial dephosphorylation of Tag with calf intestinal alkaline phosphatase stimulated replication activity in vitro (24,44) and was paralleled by an increased binding to site II (35,44,63), an essential element in the SV40 origin of replication.…”