The formation of a flexible DNA-binding protein chain is required for efficient DNA unwinding and adenovirus DNA chain elongation

The adenovirus DNA-binding protein (DBP) binds co-operatively to single-str anded DNA (ssDNA) and stimulates both initiation and elongation of DNA repl ication. DBP consists of a globular core domain and a C-terminal arm that h ooks onto a neighboring DBP molecule to form a stable protein chain with th e DNA bound to the internal surface of the chain. This multimerization is t he driving force for ATP-independent DNA unwinding by DBP during elongation . As shown by x-ray diffraction of different crystal forms of the C-termina l domain, the C-terminal arm can adopt different conformations, leading to flexibility in the protein chain. This flexibility is a function of the hin ge region, the part of the protein joining the C-terminal arm to the protei n core. To investigate the function of the flexibility, proline residues we re introduced in the hinge region, and the proteins were purified to homoge neity after baculovirus expression. The mutant proteins were still able to bind ss- and double-stranded DNA with approximately the same affinity as wi ld type, and the binding to ssDNA was found to be cooperative. All mutant p roteins were able to stimulate the initiation of DNA replication to near wi ld type levels. However, the proline mutants could not support elongation o f DNA replication efficiently. Even the elongation up to 26 nucleotides was severely impaired. This defect was also seen when DNA unwinding was studie d. Binding studies of DBP to homo-oligonucleotides showed an inability of t he proline mutants to bind to poly(dA)(40), indicating an inability to adap t to specific DNA conformations. Our data suggest that the flexibility of t he protein chain formed by DBP is important in binding and unwinding of DNA during adenovirus DNA replication. A model explaining the need for flexibi lity of the C-terminal arm is proposed.