The Formation of a Flexible DNA-binding Protein Chain Is Required for Efficient DNA Unwinding and Adenovirus DNA Chain Elongation

Abstract

The adenovirus DNA-binding protein (DBP) binds cooperatively to single-stranded DNA (ssDNA) and stimulates both initiation and elongation of DNA replication. DBP consists of a globular core domain and a C-terminal arm that hooks onto a neighboring DBP molecule to form a stable protein chain with the DNA bound to the internal surface of the chain. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. As shown by x-ray diffraction of different crystal forms of the C-terminal domain, the C-terminal arm can adopt different conformations, leading to flexibility in the protein chain. This flexibility is a function of the hinge region, the part of the protein joining the C-terminal arm to the protein core. To investigate the function of the flexibility, proline residues were introduced in the hinge region, and the proteins were purified to homogeneity after baculovirus expression. The mutant proteins were still able to bind ss-and double-stranded DNA with approximately the same affinity as wild type, and the binding to ssDNA was found to be cooperative. All mutant proteins were able to stimulate the initiation of DNA replication to near wild type levels. However, the proline mutants could not support elongation of DNA replication efficiently. Even the elongation up to 26 nucleotides was severely impaired. This defect was also seen when DNA unwinding was studied. Binding studies of DBP to homo-oligonucleotides showed an inability of the proline mutants to bind to poly(dA) 40 , indicating an inability to adapt to specific DNA conformations. Our data suggest that the flexibility of the protein chain formed by DBP is important in binding and unwinding of DNA during adenovirus DNA replication. A model explaining the need for flexibility of the C-terminal arm is proposed.

Adenovirus Type 5 DNA Binding Protein Stimulates Binding of DNA Polymerase to the Replication Origin

“…An average of three independent experiments showed that PPP-DBP stimulated initiation on TD50 is (12.3 Ϯ 1.7)-fold, and the average of DBP stimulated initiation is 13.0 Ϯ 1.5 (n ϭ 3), demonstrating that the stimulation of initiation is not affected by the PPP-DBP mutant. It should be noted that due to the slightly lower DNA binding affinity of PPP-DBP, a twofold-higher concentration is required to obtain optimal stimulation of initiation (34). Therefore, we conclude that the stimulation of initiation is independent of the unwinding activity.…”

“…In PPP-DBP three amino acids (512N, 513V, and 514S) in the hinge region of the flexible C-terminal arm are substituted by proline, thereby destroying the flexibility, which in turn results in loss of unwinding activity on TD50 and smaller dsDNA templates, whereas binding of PPP-DBP to ssDNA and dsDNA was only slightly affected (34). This mutant was still capable of stimulating initiation on the various templates (Fig.…”

“…a similar experiment with partially ssDNA was performed with an unwinding-negative DBP mutant called PPP-DBP (34). In PPP-DBP three amino acids (512N, 513V, and 514S) in the hinge region of the flexible C-terminal arm are substituted by proline, thereby destroying the flexibility, which in turn results in loss of unwinding activity on TD50 and smaller dsDNA templates, whereas binding of PPP-DBP to ssDNA and dsDNA was only slightly affected (34).…”

“…The adenovirus proteins ⌬N-DBP, PPP-DBP, Pol, Pol exo mutant (D422A), and pTP were expressed in baculovirus-infected Sf9 cells and purified to near homogeneity, as verified by silver staining as described previously (5,6,9,34). Phage T4 gp32 and T4 Pol were purchased from Amersham Pharmacia Biotech.…”

“…Deletion of the C-terminal arm results in a loss of cooperative DBP binding to ssDNA and also abolishes the DNA unwinding activity of DBP (11). Recently, we determined that the flexibility of the DBP molecule is important for the DNA unwinding activity of DBP as it functions to adapt the ssDNA and dsDNA structures present at the replication fork during initiation (34).…”