Role of Base Flipping in Specific Recognition of Damaged DNA by Repair Enzymes

doi:10.1016/S0022-2836(02)00999-3

Journal of Molecular Biology

Volume 323, Issue 5, 8 November 2002, Pages 823-834

https://doi.org/10.1016/S0022-2836(02)00999-3 Get rights and content

Abstract

DNA repair enzymes induce base flipping in the process of damage recognition. Endonuclease V initiates the repair of cis, syn thymine dimers (TD) produced in DNA by UV radiation. The enzyme is known to flip the base opposite the damage into a non-specific binding pocket inside the protein. Uracil DNA glycosylase removes a uracil base from G·U mismatches in DNA by initially flipping it into a highly specific pocket in the enzyme. The contribution of base flipping to specific recognition has been studied by molecular dynamics simulations on the closed and open states of undamaged and damaged models of DNA. Analysis of the distributions of bending and opening angles indicates that enhanced base flipping originates in increased flexibility of the damaged DNA and the lowering of the energy difference between the closed and open states. The increased flexibility of the damaged DNA gives rise to a DNA more susceptible to distortions induced by the enzyme, which lowers the barrier for base flipping. The free energy profile of the base-flipping process was constructed using a potential of mean force representation. The barrier for TD-containing DNA is 2.5 kcal mol⁻¹ lower than that in the undamaged DNA, while the barrier for uracil flipping is 11.6 kcal mol⁻¹ lower than the barrier for flipping a cytosine base in the undamaged DNA. The final barriers for base flipping are approximately 10 kcal mol⁻¹, making the rate of base flipping similar to the rate of linear scanning of proteins on DNA. These results suggest that damage recognition based on lowering the barrier for base flipping can provide a general mechanism for other DNA-repair enzymes.

Introduction

Accumulating evidence points to the importance of base flipping in DNA damage recognition by repair enzymes.1., 2., 3., 4., 5., 6., 7., 8., 9. However, the underlying mechanism of base flipping is not clearly understood.6., 10., 11., 12., 13. All the presently known structures of repair enzymes in complex with damaged DNA exhibit a flipped-out base accompanied by substantial bending and distortion of the DNA. It has been proposed that bending and twisting of DNA facilitates base-pair opening.14., 15. The static analysis demonstrated that base opening required less energy in bent or unwound DNA than in its canonical form. However, the nature of the dynamic coupling between DNA distortion and opening cannot be obtained from these studies. Recent free-energy simulations of base-pair flipping¹⁶ illustrate the complex nature of the opening process, but do not provide information on the coupling between opening and other distortions. Essential dynamic analysis of molecular dynamics (MD) simulations of undamaged DNA and DNA with a thymine dimer (TD) suggested a specific correlation between bending and opening motions of the adenine base complementary to the 5′ thymine base of TD, which was missing in the undamaged DNA.¹⁷ It therefore appears that the nature of the coupling between bending and base flipping is dynamic and depends on specific local flexibility properties of the DNA.

Cis,syn TD is one of the major photoproducts of the interaction of UV radiation with DNA. The presence of TD in DNA is a major obstacle in DNA replication, and is therefore potentially lethal. Repair of the TD lesion is essential for maintaining proper function of DNA. Endonuclease V (endoV) has been shown to initiate repair of the damaged DNA by excising the TD efficiently.¹⁸ The catalytic steps in the enzymatic action of endoV are fairly well understood19., 20. but the highly specific recognition of TD remains unclear.²¹ Examination of the structures of the free enzyme²² and of the complex with a TD-containing DNA²³ demonstrates that the protein remains nearly unchanged while the major structural changes are induced in the DNA. However, in spite of extensive experimental and theoretical studies on TD-containing DNA sequences,24., 25., 26., 27., 28., 29. the properties of the damaged sequence that are recognized specifically by endoV have not been elucidated unequivocally.

The TD induces only local perturbations in the structure of the damaged DNA characterized by changes in inter base-pair parameters24., 28., 29. and impaired base-pairing between the 5′ TD and the opposite adenine base as reflected in a longer hydrogen bond.17., 26., 28., 30. These changes are produced mostly by the steric restraint imposed on the DNA helix by the cyclobutane ring of the TD.¹⁷ These distortions, as well as those observed by NMR,²⁷ cannot account for specific recognition because the crystal structure of the TD-containing DNA in complex with endoV²³ deviates significantly from the structure in solution. It appears that specificity of damage recognition by the enzyme does not localize to a single static property of the damaged DNA.

In the crystal structure of the complex,²³ the DNA exhibits three remarkable structural changes induced by the protein. The DNA in the complex is bent significantly at the position of the TD and exhibits a large helical twist in the vicinity of the kink. Furthermore, the adenine base opposite the 5′-thymine base of the TD is flipped out and inserted into a binding pocket without forming specific hydrogen bonds with the protein residues. The space left by the flipped-out base provides access to the site of damage by the catalytic residues. The only direct contact between the protein and the lesion is a hydrogen bond between Arg26 and the O₍₂₎ of the 5′-thymine base in TD and several contacts between the protein and the phosphate groups are mediated through water molecules. Thus, the authors²³ propose that the recognition of the DNA with TD is accomplished through an indirect readout and the kink induced in the DNA by the protein is coupled to the flipping of the adenine base opposite the TD. Nevertheless, the details of the coupling between bending and flipping remain unclear and the structure fails to explain the essential difference between a damaged and a native DNA.

Uracil is a base-damage in DNA that originates through misincorporation or hydrolytic deamination of cytosine.³¹ Uracil DNA glycosylase (UDG) is a very efficient repair enzyme, which has been studied extensively by experimental and theoretical methods.32., 33., 34., 35., 36. Crystal structures of UDG with a uracil-containing DNA show that the uracil base is flipped out and anchored in a specific pocket inside the enzyme.7., 37., 38. The structure of the DNA is distorted substantially with a kink of 45° near the flipped uracil base. Recent studies of the contributions of various residues of UDG to the formation of the complex with the DNA identified three distinct stages in complex formation.39., 40. An early step consists of pinching the phosphodiester backbone followed by a pushing and plugging step. Both steps seem to enhance the base flipping and stabilize the flipped-out state. The last stage consists of pulling by the residues in the active-site pocket, which is associated with a conformational change of the UDG. These studies and the comparison of the distortions in the complexed DNA structure to those induced by a G·U mismatch⁴¹ illustrate again that the minor structural changes produced by a wobble base-pair cannot account for the specificity of damage recognition. Thus, in order to understand the role the proteins play in enhancing base flipping as part of their enzymatic specificity, we need to formulate a link between the damage in DNA and its flexibility properties that enhance spontaneous base flipping.

In the present work, we use MD simulations to construct a potential of mean force (PMF) representation of the free-energy profile of bending and base opening. We find that the damage enhances the coupling between bending and base opening, which lowers the barrier for base flipping in the damaged sequences. Thus, the dynamic properties of DNA that influence the interaction between the repair enzyme and the DNA play an important role in specific damage recognition.

Section snippets

Results

We have conducted 1.5 ns simulations of sequences with a TD and for comparison with a TT that represents an undamaged DNA with the same sequence. To investigate the effect of a G·U mismatch in comparison to a normal G·C-containing DNA, we have conducted similar simulations of the sequences shown in Table 1. To assess the stability of the simulations the RMS deviations of all atoms from their average positions have been calculated as a function of time. They fluctuate around a nearly constant

Discussion

In this work, we have analyzed the effect of two forms of DNA damage, a TD and a G·U mismatch, on the bending and opening properties of damaged DNA. Through a PMF representation of the two motions in the closed and open states of the same sequence we were able to combine results from separate simulations and provide an energetic description of the process. The results show that while different types of damage reduce the activation energy of base opening by different amount, both bring the

Starting structures

Sequences used for MD simulations are displayed in Table 1. Initial structures of the undamaged models (sequences I, II, V and VI) have been constructed with standard B-DNA geometries. The TD was incorporated in sequences III and IV to replace the T6/T7 step. Conformation and atomic charges of the TD have been determined.²⁸ The DNA with an extrahelical Ade19 was adopted from the crystal structure of the endoV–DNA complex¹¹ by deleting the protein. The initial structure of model VII was

Acknowledgements

This work was supported, in part, by US PHS grant CA 63317. The research has been sponsored by OTKA grants T30566, T34131 as well as Bolyai and OTKA D34572 fellowships.

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Journal of Molecular Biology

Role of Base Flipping in Specific Recognition of Damaged DNA by Repair Enzymes

Abstract

Introduction

Section snippets

Results

Discussion

Starting structures

Acknowledgements

Curr. Opin. Biotechnol.

J. Biol. Chem.

Structure

Cell

J. Biol. Chem.

Structure

Chem. Biol.

J. Mol. Struct. (THEOCHEM)

Mutat. Res.

J. Mol. Biol.

Mutat. Res.

J. Mol. Biol.

Cell

J. Mol. Biol.

Prog. Nucl. Acid Res. Mol. Biol.

Prog. Nucl. Acid Res. Mol. Biol.

Arch. Biochem. Biophys.

J. Mol. Biol.

Cell

Base flipping

Annu. Rev. Biochem.

DNA glycosylases in the base excision repair of DNA

Biochem. J.

Mechanistic link between DNA methyltransferases and DNA repair enzymes by base flipping

Biopolymers

A nucleotide flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA

Nature

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases

Nucl. Acids Res.