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Template melting is an essential step in the initiation of DNA replication, but the mechanism of template melting is unknown for any replicon. Here we demonstrate that melting of the bovine papillomavirus type 1 ori is a sequence-dependent process which relies on specific recognition of TA base pairs in the minor groove by the E1 initiator. We show that correct template melting is a prerequisite for the formation of a stable double hexamer with helicase activity and that ori mutants that fail to melt correctly are defective for ori unwinding and DNA replication in vivo. Our results also indicate that melting of the DNA is achieved by destabilization of the double helix along its length through multiple interactions with E1, each of which is responsible for melting of a few base pairs, resulting in the extensive melting that is required for initiation of DNA replication.

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein