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

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

Titration of 20bp and 28bp DNAs with human AGT, detection by EMSA. Upper panel: 20‐mer duplex DNA, 1.35 × 10−9 M with [AGT] increasing from 0 M to 3.5 × 10−6 M from left to right. Lower panel: 28‐mer duplex DNA, 3.6 × 10−9 M, with [AGT] increasing from 0 M to 5.5 × 10−6 M from left to right. Binding reactions were carried out at 20°C ± 1°C and samples were resolved on 10% polyacrylamide gels, as described in Experimental Procedures. Band designations B, bound DNA; F, free DNA.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016775&req=5

bip22684-fig-0002: Titration of 20bp and 28bp DNAs with human AGT, detection by EMSA. Upper panel: 20‐mer duplex DNA, 1.35 × 10−9 M with [AGT] increasing from 0 M to 3.5 × 10−6 M from left to right. Lower panel: 28‐mer duplex DNA, 3.6 × 10−9 M, with [AGT] increasing from 0 M to 5.5 × 10−6 M from left to right. Binding reactions were carried out at 20°C ± 1°C and samples were resolved on 10% polyacrylamide gels, as described in Experimental Procedures. Band designations B, bound DNA; F, free DNA.

Mentions: Titration of short DNAs with AGT gave mixtures containing the free duplex and a saturated protein‐DNA complex. Mobility‐shift profiles for mixtures containing the 20bp and 28bp DNAs are shown in Figure 2. Global analyses of sedimentation equilibirum data obtained under conditions of AGT‐excess (Figure 3) returned Mr(complex, 20mer) = 115,026 ± 4,235 and Mr(complex, 28mer) = 170,466 ± 5,138, respectively, consistent with AGT stoichiometries of 4.8 ± 0.17 for the 20mer and 7.11 ± 0.21 for the 28mer. These stiochiometries are consistent with binding site sizes of ∼4bp/protein found for dsDNAs of other lengths40, 53 and they conform to the model shown in Figure 1, in which protein molecules are tightly packed along the DNA contour.


Resolving the contributions of two cooperative mechanisms to the DNA Binding of AGT
Titration of 20bp and 28bp DNAs with human AGT, detection by EMSA. Upper panel: 20‐mer duplex DNA, 1.35 × 10−9 M with [AGT] increasing from 0 M to 3.5 × 10−6 M from left to right. Lower panel: 28‐mer duplex DNA, 3.6 × 10−9 M, with [AGT] increasing from 0 M to 5.5 × 10−6 M from left to right. Binding reactions were carried out at 20°C ± 1°C and samples were resolved on 10% polyacrylamide gels, as described in Experimental Procedures. Band designations B, bound DNA; F, free DNA.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016775&req=5

bip22684-fig-0002: Titration of 20bp and 28bp DNAs with human AGT, detection by EMSA. Upper panel: 20‐mer duplex DNA, 1.35 × 10−9 M with [AGT] increasing from 0 M to 3.5 × 10−6 M from left to right. Lower panel: 28‐mer duplex DNA, 3.6 × 10−9 M, with [AGT] increasing from 0 M to 5.5 × 10−6 M from left to right. Binding reactions were carried out at 20°C ± 1°C and samples were resolved on 10% polyacrylamide gels, as described in Experimental Procedures. Band designations B, bound DNA; F, free DNA.
Mentions: Titration of short DNAs with AGT gave mixtures containing the free duplex and a saturated protein‐DNA complex. Mobility‐shift profiles for mixtures containing the 20bp and 28bp DNAs are shown in Figure 2. Global analyses of sedimentation equilibirum data obtained under conditions of AGT‐excess (Figure 3) returned Mr(complex, 20mer) = 115,026 ± 4,235 and Mr(complex, 28mer) = 170,466 ± 5,138, respectively, consistent with AGT stoichiometries of 4.8 ± 0.17 for the 20mer and 7.11 ± 0.21 for the 28mer. These stiochiometries are consistent with binding site sizes of ∼4bp/protein found for dsDNAs of other lengths40, 53 and they conform to the model shown in Figure 1, in which protein molecules are tightly packed along the DNA contour.

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