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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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Dependence of K and ω on DNA length. Points designated with arrows are results of experiments shown in Figure 4. Data for other DNA lengths are from Melikishvili et al.40 The error bars correspond to 95% confidence limits estimated for each parameter. The points corresponding to K‐values for dA24‐dT24 and dG24‐dC24 templates are labeled. The smooth curve fitting the dependence of ω on N is the sum of a baseline with a constant, positive slope and a cosine function40 with a period of 4.02 ± 0.05 bp/cycle.
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bip22684-fig-0005: Dependence of K and ω on DNA length. Points designated with arrows are results of experiments shown in Figure 4. Data for other DNA lengths are from Melikishvili et al.40 The error bars correspond to 95% confidence limits estimated for each parameter. The points corresponding to K‐values for dA24‐dT24 and dG24‐dC24 templates are labeled. The smooth curve fitting the dependence of ω on N is the sum of a baseline with a constant, positive slope and a cosine function40 with a period of 4.02 ± 0.05 bp/cycle.

Mentions: Equation (1) resolves overall binding affinity into ensemble‐average values of K and ω. Shown in Figure 4 are Scatchard plots of data for AGT binding to 20bp and 28bp duplexes. Fitting the data with Eq. (1) returned values of K(ave, 20mer) = 8286 ± 1134 M−1, ω(ave, 20mer) = 112.1± 18.1, and K(ave, 28mer) = 10174 ± 1081 M−1, ω(ave, 28mer) = 127.9 ± 13.6. These values are comparable to others found under the same conditions of temperature and buffer composition, but using different DNAs (Figure 5). Values of K and ω oscillate with increasing DNA length, and the fact that maxima for K and ω occur at both 16bp and 20bp DNA lengths suggests that protein‐DNA and protein‐protein interactions may be coupled to some degree (In a study of drug‐DNA interactions, Correia and Chaires found values of K and ω were strongly correlated 54; this remains an important caveat in the use of least‐squares fitting using the McGhee‐von Hippel equation and related expressions). However, it is clear that DNA sequence, or possibly base composition, also has an effect on K (compare values for dG24•dC24 and dA24•dT24 substrates) and sequence effects on binding to the rest of the heterogeneous DNA population may obscure periodicity in K as DNA length increases.


Resolving the contributions of two cooperative mechanisms to the DNA Binding of AGT
Dependence of K and ω on DNA length. Points designated with arrows are results of experiments shown in Figure 4. Data for other DNA lengths are from Melikishvili et al.40 The error bars correspond to 95% confidence limits estimated for each parameter. The points corresponding to K‐values for dA24‐dT24 and dG24‐dC24 templates are labeled. The smooth curve fitting the dependence of ω on N is the sum of a baseline with a constant, positive slope and a cosine function40 with a period of 4.02 ± 0.05 bp/cycle.
© Copyright Policy - creativeCommonsBy-nc-nd
Related In: Results  -  Collection

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

bip22684-fig-0005: Dependence of K and ω on DNA length. Points designated with arrows are results of experiments shown in Figure 4. Data for other DNA lengths are from Melikishvili et al.40 The error bars correspond to 95% confidence limits estimated for each parameter. The points corresponding to K‐values for dA24‐dT24 and dG24‐dC24 templates are labeled. The smooth curve fitting the dependence of ω on N is the sum of a baseline with a constant, positive slope and a cosine function40 with a period of 4.02 ± 0.05 bp/cycle.
Mentions: Equation (1) resolves overall binding affinity into ensemble‐average values of K and ω. Shown in Figure 4 are Scatchard plots of data for AGT binding to 20bp and 28bp duplexes. Fitting the data with Eq. (1) returned values of K(ave, 20mer) = 8286 ± 1134 M−1, ω(ave, 20mer) = 112.1± 18.1, and K(ave, 28mer) = 10174 ± 1081 M−1, ω(ave, 28mer) = 127.9 ± 13.6. These values are comparable to others found under the same conditions of temperature and buffer composition, but using different DNAs (Figure 5). Values of K and ω oscillate with increasing DNA length, and the fact that maxima for K and ω occur at both 16bp and 20bp DNA lengths suggests that protein‐DNA and protein‐protein interactions may be coupled to some degree (In a study of drug‐DNA interactions, Correia and Chaires found values of K and ω were strongly correlated 54; this remains an important caveat in the use of least‐squares fitting using the McGhee‐von Hippel equation and related expressions). However, it is clear that DNA sequence, or possibly base composition, also has an effect on K (compare values for dG24•dC24 and dA24•dT24 substrates) and sequence effects on binding to the rest of the heterogeneous DNA population may obscure periodicity in K as DNA length increases.

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