Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence

Kuil, Mark; Amerongen, Herbert van; Vliet, Peter C. van der; Grondelle, Rienk van

doi:10.1021/bi00451a038

Cited by 27 publications

(19 citation statements)

References 34 publications

(50 reference statements)

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“…For instance, since the number of overlapping binding sites on a DNA lattice is given by N Ϫ m ϩ 1, the 75-bp duplex would have 51 potential overlapping binding sites assuming that m ϭ 25. Previous studies have already shown the importance of applying an overlapping model to appropriately describe the interaction of nonspecific DNA-binding proteins with linear DNA lattices (16,27,35,36). On the other hand, the possibility that the binding sites might overlap was never properly considered in the case of the Ku-DNA interaction.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence Anisotropy Studies on the Ku-DNA Interaction

Arosio

Costantini

Kong

et al. 2004

Journal of Biological Chemistry

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DNA non-homologous end joining starts with the binding of Ku heterodimers to double strand breaks. In this work, we characterized the thermodynamics of the Ku-DNA interaction by fluorescence anisotropy of the probe-labeled DNA. We determined that the microscopic dissociation constant (k d ) for the binding of Ku to a DNA binding site of the proper length (>20 bp) ranges from 22 to 29 nM at 300 mM NaCl. The binding isotherms for DNA duplexes with two or three heterodimers were analyzed with two independent models considering the presence and absence of overlapping binding sites. This analysis demonstrated that there is no or very weak nearestneighbor cooperativity among the Ku molecules. These models can most likely be applied to study the interaction of Ku with duplexes of any length. Furthermore, our salt dependence studies indicated that electrostatic interactions play a major role in the binding of Ku to DNA and that the k d decreases ϳ60-fold as the salt concentration is lowered from 300 to 200 mM. The slope (⌫ salt ) of the plot of log k d versus log[NaCl] is 12.4 ؎ 0.1. This value is among the highest reported in the literature for a protein-DNA interaction and suggests that ϳ12 ions are released upon formation of the Ku-DNA complex. In addition, comparison of the slope values measured upon varying the type of cation and anion indicated that approximately nine cations and three anions are released from DNA and Ku, respectively, when the complex is formed.

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Section: Discussionmentioning

confidence: 99%

Fluorescence Anisotropy Studies on the Ku-DNA Interaction

Arosio

Costantini

Kong

et al. 2004

Journal of Biological Chemistry

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“…However, we have not studied the stoichiometry of the bands in detail. The lack of cooperativity also leads to a lower binding affinity that is reflected by the higher concentration of proteins needed to obtain a shifted complex (35).…”

Section: The Hinge Mutants Bind Ss-and Dsdna With Approximatelymentioning

confidence: 99%

“…This might be reflected by the problems in binding poly(dA) which has an aberrant structure and a different binding site for DBP (30,35).…”

Section: Reduction Of the Flexibility Of The C-terminal Arm Does Not mentioning

confidence: 99%

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

Breukelen¹,

Kanellopoulos²,

Tucker³

et al. 2000

Journal of Biological Chemistry

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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.

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“…DBP binds cooperatively to single-stranded DNA (ssDNA) with a binding site size of ca. 12 nucleotides in a sequence-independent manner (21). Mild chymotrypsin treatment cleaves DBP into two domains: an N-terminal part (aa 1 to 173) and a C-terminal part (aa 174 to 529) that is well conserved among different serotypes and harbors most of the biological functions ascribed to DBP, including its nucleic acidbinding and replication functions (32).…”

mentioning

confidence: 99%

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

Breukelen

Brenkman

Holthuizen

et al. 2003

J Virol

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The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K m for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.

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Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence

Cited by 27 publications

References 34 publications

Fluorescence Anisotropy Studies on the Ku-DNA Interaction

Fluorescence Anisotropy Studies on the Ku-DNA Interaction

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

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

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