Topography of simian virus 40 A protein-DNA complexes: arrangement of protein bound to the origin of replication

Abstract

DNA binding regions I, II, and III at the origin of replication have different arrangements of A protein (T antigen) recognition pentanucleotides. The A protein also protects each region from DNase in distinctly different patterns. Footprint and fragment assays led to the following conclusions: (i) in some cases a single recognition pentanucleotide is sufficient to direct the binding and accurate alignment of A protein on DNA; (ii) the A protein binds within isolated region I or II in a sequential process leading to multiple overlapping areas of DNase protection within each region; and (iii) the 23-base pair span of recognition sequences in region II allows binding and protection of a longer length of DNA than the 23-base pair span in region I. We propose a model of protein binding that addresses the problem of variations in the arrangement of pentanucleotides in regions I and II and explains the observed DNase protection patterns. The central feature of the model requires each protomer of A protein to bind to a pentanucleotide in a unique direction. The resulting orientation of protein would protect more DNA at the 5' end of the 5'-GAGGC-3' recognition sequence than at the 3' end. The arrangement of multiple protomers at the origin of simian virus 40 replication is discussed. of PIPES binding buffer with 0.1 M MgCl2 and 0.05 M CaCl2 was added for 5 min at 0°C in 0.005 M NaCl.

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

“…These experiments indicate that T-ag-bd 131-260 molecules bound to site I do not stimulate the binding of T-ag-bd 131-260 molecules to site II. This observation is consistent with previous studies of T-ag which indicated that there was little or no cooperativity between T-ag molecules bound to these two sites (7,83). Finally, the experiments presented in Fig.…”

“…1B) nucleate T-ag hexamer assembly (59). Related experiments indicated that pentanucleotide 1 is the strongest binding site in the core origin palindrome (12,40,83). In view of these observations, the band shift assays were initially conducted with T-ag-bd 131-260 and oligonucleotides containing transition mutations in either pentanucleotide 1 or 2.…”

Critical spatial requirement within the origin of simian virus 40 DNA replication

“…The 0 at the top of a gel well indicates that no A protein was added to the DNA. tide 1 is the strongest binding site in the origin palindrome (17,28). We have emphasized this feature in the summary of DMS protection results in Fig.…”

“…Subunits of A protein bound to each pentanucleotide in a specific orientation that protected ca. 20 bp at the 3' end of the recognition sequence and 10 bp at the 5' end (28). These DNase protection domains are shown by the arrows in The HindIII-Ncol origin fragment was labeled on the 3' HindIl end and exposed to DNase I in the presence or absence of A protein at two NaCl concentrations.…”

“…Bound and protected DNA was selected on nitrocellulose filters, eluted, and analyzed in nondenaturing 12% polyacrylamide gels. The map locations of protected fragments relative to recognition pentanucleotides and DNase footprint regions of the origin are shown at the bottom of the figure (28). The 60to 65-bp fragment 2 corresponds to the sequence shown in Fig.…”