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

Abstract

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.

Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin

“…Since the [α-32 P]-DNA strands used in this hybridization can be chased into higher molecular weight intermediates, we conclude that the administration of 2-AP prior to the ODP disrupts the pattern of initiation of genuine replication forks. Although the detection of alternative start sites for replication required transformation with SV40, it is unlikely that their recognition is due to the initiation activity of the viral T antigen protein since: (i) initiation at apparently random sites required the administration of 2-AP prior to the ODP; (ii) Chinese hamster cells are not permissive for SV40 replication (Gilbert and Cohen, 1990); and (iii) stable binding of SV40 T antigen to DNA requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix, and positioned in a head-to-head orientation (Joo et al, 1997), a configuration that is unlikely to occur at all in the CHO genome. A more likely interpretation of Fig.…”

Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA

“…The N-terminal region (J-domain) is not required for in vitro DNA replication (Chen et al, 1997;Kim et al, 2001). The origin-binding domain (OBD) maps to amino acids 131-259 (Arthur et al, 1988;Joo et al, 1997). Mutagenesis studies of this domain in the context of the full-length protein also suggest a role for this domain in oligomerization and DNA structural perturbations.…”

Characterization of the DNA-Binding Properties of the Origin-Binding Domain of Simian Virus 40 Large T Antigen by Fluorescence Anisotropy

“…These two essential activities map to different domains of these proteins. For SV40 large T antigen and papillomavirus E1, a minimal sequence-specific DNA binding domain has been identified in the middle of the protein, N-terminal to the ATPase/helicase domain (5,15,23,17,27). The minimal origin-binding domain (OBD) of SV40 large T antigen, located between amino acids 131 and 260 ( Fig.…”

“…It is structurally similar to the minimal DNA-binding domain (DBD) of the bovine papillomavirus E1 protein despite the fact that both domains share only 6% identity at the primary amino acid sequence level (10). The T-antigen OBD and E1 DBD are monomeric proteins in solution but bind DNA preferentially as dimers to pairs of inverted binding sites in electrophoretic mobility shift assays (EMSA) (5,15,19). The fact that EMSA are not performed at true equilibrium and are not suitable to measure weak interactions prompted us to reexamine the binding of the SV40 T antigen OBD to its origin using an assay based on fluorescence anisotropy (13,18).…”