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Resolving the contributions of two cooperative mechanisms to the DNA Binding of AGT

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ABSTRACT

The O6‐alkylguanine DNA alkyltransferase (AGT) is a DNA repair enzyme that binds DNA with moderate cooperativity. This cooperativity is important for its search for alkylated bases. A structural model of the cooperative complex of AGT with DNA predicts short‐range interactions between nearest protein neighbors and long‐range interactions between proteins separated in the array. DNA substrates ranging from 11bp to 30bp allowed us to use differences in binding stoichiometry to resolve short‐ and long‐range protein contributions to the stability of AGT complexes. We found that the short‐range component of ΔG°coop was nearly independent of DNA length and protein packing density. In contrast the long‐range component oscillated with DNA length, with a period equal to the occluded binding site size (4bp). The amplitude of the long‐range component decayed from ∼−4 kcal/mole of interaction to ∼−1.2 kcal/mol of interaction as the size of cooperative unit increased from 4 to 7 proteins, suggesting a mechanism to limit the size of cooperative clusters. These features allow us to make testable predictions about AGT distributions and interactions with chromatin structures in vivo. © 2015 The Authors Biopolymers Published by Wiley Periodicals, Inc. Biopolymers 103: 509–516, 2015.

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Model of a cooperative AGT‐DNA complex formed on double‐stranded DNA. The repeating unit of this model is one molecule of AGT (colors) plus 4 base‐pairs of DNA (black); the coordinates were derived from the crystal structure of Daniels et al.37 Repeating units were juxtaposed with preservation of B‐DNA helical parameters (separation = 3.4 Å, twist = 34.6°) between base‐pairs of adjacent units. For details of the construction of this model and data supporting it, see Adams et al.42 Left panel: side view with N‐terminal faces of proteins oriented to left. Right panel: end‐view showing the C‐terminal faces of proteins.
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bip22684-fig-0001: Model of a cooperative AGT‐DNA complex formed on double‐stranded DNA. The repeating unit of this model is one molecule of AGT (colors) plus 4 base‐pairs of DNA (black); the coordinates were derived from the crystal structure of Daniels et al.37 Repeating units were juxtaposed with preservation of B‐DNA helical parameters (separation = 3.4 Å, twist = 34.6°) between base‐pairs of adjacent units. For details of the construction of this model and data supporting it, see Adams et al.42 Left panel: side view with N‐terminal faces of proteins oriented to left. Right panel: end‐view showing the C‐terminal faces of proteins.

Mentions: Human AGT is a well‐characterized protein31, 34, 37, 38 that binds undamaged DNA with little sequence‐specificity or base‐composition‐specificity and only moderate cooperativity.10, 39, 40 It binds the minor groove face of B‐form DNA, bending the DNA toward the major groove by ∼15 degrees37 and unwinding the DNA by ∼7 degrees.41 It occupies ∼8bp along one face of the DNA cylinder,37 but each molecule excludes others from only 4bp40; thus protein molecules overlap along the DNA contour. At binding saturation on torsionally‐relaxed DNA, this produces a three‐start helical array of proteins with each monomer displaced by ∼13.6 Å along the helix axis and rotated ∼138 degrees with respect to its nearest neighbors42 (Figure 1). This combination of rotation and translation allows little contact between proteins that are nearest neighbors, but proteins in relative positions n and n+3 make extensive contact (ΔASA ∼1100Å2).42 Accordingly, we predicted that the n‐to‐n+3 contact would contribute more to binding cooperativity than would nearest neighbor interactions. Here, we use a combination of new data and data from a previous study,40 together with a new analysis strategy, to test this prediction.


Resolving the contributions of two cooperative mechanisms to the DNA Binding of AGT
Model of a cooperative AGT‐DNA complex formed on double‐stranded DNA. The repeating unit of this model is one molecule of AGT (colors) plus 4 base‐pairs of DNA (black); the coordinates were derived from the crystal structure of Daniels et al.37 Repeating units were juxtaposed with preservation of B‐DNA helical parameters (separation = 3.4 Å, twist = 34.6°) between base‐pairs of adjacent units. For details of the construction of this model and data supporting it, see Adams et al.42 Left panel: side view with N‐terminal faces of proteins oriented to left. Right panel: end‐view showing the C‐terminal faces of proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016775&req=5

bip22684-fig-0001: Model of a cooperative AGT‐DNA complex formed on double‐stranded DNA. The repeating unit of this model is one molecule of AGT (colors) plus 4 base‐pairs of DNA (black); the coordinates were derived from the crystal structure of Daniels et al.37 Repeating units were juxtaposed with preservation of B‐DNA helical parameters (separation = 3.4 Å, twist = 34.6°) between base‐pairs of adjacent units. For details of the construction of this model and data supporting it, see Adams et al.42 Left panel: side view with N‐terminal faces of proteins oriented to left. Right panel: end‐view showing the C‐terminal faces of proteins.
Mentions: Human AGT is a well‐characterized protein31, 34, 37, 38 that binds undamaged DNA with little sequence‐specificity or base‐composition‐specificity and only moderate cooperativity.10, 39, 40 It binds the minor groove face of B‐form DNA, bending the DNA toward the major groove by ∼15 degrees37 and unwinding the DNA by ∼7 degrees.41 It occupies ∼8bp along one face of the DNA cylinder,37 but each molecule excludes others from only 4bp40; thus protein molecules overlap along the DNA contour. At binding saturation on torsionally‐relaxed DNA, this produces a three‐start helical array of proteins with each monomer displaced by ∼13.6 Å along the helix axis and rotated ∼138 degrees with respect to its nearest neighbors42 (Figure 1). This combination of rotation and translation allows little contact between proteins that are nearest neighbors, but proteins in relative positions n and n+3 make extensive contact (ΔASA ∼1100Å2).42 Accordingly, we predicted that the n‐to‐n+3 contact would contribute more to binding cooperativity than would nearest neighbor interactions. Here, we use a combination of new data and data from a previous study,40 together with a new analysis strategy, to test this prediction.

View Article: PubMed Central - PubMed

ABSTRACT

The O6‐alkylguanine DNA alkyltransferase (AGT) is a DNA repair enzyme that binds DNA with moderate cooperativity. This cooperativity is important for its search for alkylated bases. A structural model of the cooperative complex of AGT with DNA predicts short‐range interactions between nearest protein neighbors and long‐range interactions between proteins separated in the array. DNA substrates ranging from 11bp to 30bp allowed us to use differences in binding stoichiometry to resolve short‐ and long‐range protein contributions to the stability of AGT complexes. We found that the short‐range component of ΔG°coop was nearly independent of DNA length and protein packing density. In contrast the long‐range component oscillated with DNA length, with a period equal to the occluded binding site size (4bp). The amplitude of the long‐range component decayed from ∼−4 kcal/mole of interaction to ∼−1.2 kcal/mol of interaction as the size of cooperative unit increased from 4 to 7 proteins, suggesting a mechanism to limit the size of cooperative clusters. These features allow us to make testable predictions about AGT distributions and interactions with chromatin structures in vivo. © 2015 The Authors Biopolymers Published by Wiley Periodicals, Inc. Biopolymers 103: 509–516, 2015.

No MeSH data available.


Related in: MedlinePlus