The simian virus 40 origin of replication contains a 20-base-pair adenine-thymine-rich segment with the sequence 5'-TGCATAAATAAAAAAAATTA-3'. The continuous tract of eight adenines is highly conserved among polyomaviruses. We used single-base substitutions to map structural and functional features of this DNA. Mutations in the AAA and AAAAAAAATT sequences significantly reduce DNA replication and thus identify two sequence-specific functional domains or a single domain with two parts. The AAAAAAAATT sequence also determines a DNA conformation that is characteristic of DNA bending. Single-base mutations in this domain change the degree of net bending, presumably by altering the length and location of the bending sequence. Thus, DNA bending in the correct conformation and location may be a structural signal for replication in polyomavirus origins and perhaps in other origins of replication with consecutive runs of adenines. The first five base pairs (TGCAT) of the 20-base-pair segment and the T between the AAA and AAAAAAAATT domains serve a sequence-independent function that may establish proper spacing within the core origin.Adenine-thymine (AT) tracts are a common feature of a wide variety of replication origins in both procaryotes and eucaryotes (7,12,13,20,23,26). The relatively weak base pairing of these tracts would facilitate strand separation, an early and indispensable step in the initiation of DNA replication. The AT segments also contain continuous runs of adenine residues of various lengths. These runs may encode an additional signal common to the initiation process. Replication origins of polyomaviruses conform to this general sequence pattern (4). The viruses control their own replication through the interaction of the virus-encoded T antigen with a distinct viral origin of replication. Immediately adjacent to the T-antigen binding site is an AT segment of 15 to 20 base pairs (bp) (4). The AT segment of each virus contains a continuous run of at least six adenine residues at an equivalent position relative to the T-antigen recognition site. Thus, this viral AT domain provides an excellent opportunity to probe the importance and nature of adenine tracts in DNA replication. Because polyomaviruses are highly dependent on host replication factors, their AT segments may resemble components of cellular origins.The origin of the polyomavirus simian virus 40 (SV40) consists of multiple distinct elements. Although flanking regulatory regions of the early promoter-operator facilitate replication, an essential core origin is sufficient to initiate replication (6,15,19). We have shown that the core origin consists of nucleotides 5211 through 31 (4). These 64 bp contain multiple functional domains with strict sequence requirements and spacer regions with relaxed sequence specificity but precise positional constraints. The central domain is the T-antigen binding site (5, 21). We have recently mapped a 10-bp sequence-specific, functional domain and a 9-bp spacer DNA to the early side of the T-antigen binding site (4)...
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