Interactions of the DNA Repair Enzyme Human Thymine DNA

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Interactions of the DNA Repair Enzyme Human Thymine DNA Glycosylase with cognate and non-cognate DNA Natalia Kanaan, and Petra Imhof Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00409 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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Biochemistry

Interactions of the DNA Repair Enzyme Human Thymine DNA Glycosylase with cognate and non-cognate DNA Natalia Kanaan

‡Institute

‡,§

∗,‡

and Petra Imhof

of Theoretical Physics, Free University Berlin, Arnimallee 14,D-14195 Berlin, Germany

§present

address: Institut Torrent dels Alous Passatge Marconi 13-15 08191, RUBÍ (BARCELONA), Spain

E-mail: [email protected]

Running header Interactions of the DNA Repair Enzyme Human Thymine DNA Glycosylase with cognate and non-cognate DNA

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Abstract

Glycosylases specically recognise and ip their target base out of the DNA helix into the enzyme's active site. Our simulations show that a partially ipped state, already present in free DNA carrying a T:G mispair, becomes the more probable state compared to the closed stated after binding of thymine DNA glycosylase (TDG). Paired thymine (T:A) or methyl-cytosine (mC:G) do not exhibit a partially ipped state in free or complexed DNA. Important enzyme-DNA interactions exhibit signicant strength in the intra-helical and extra-helical TDG-DNA complexes. The computed binding free energy dierences suggest these interactions to account for the stabilisation of the partially ipped state, thereby driving the T:G mispair towards base ip. In the fully ipped state, the cognate base thymine is signicantly better accommodated in the enzyme's active site than non-cognate bases, suggesting the hydrolysis step as the last of several stages at which base recognition can be achieved.

Introduction DNA damage such as deamination of cytosine or methyl-cytosine results in a mismatch in the DNA with U:G or T:G mispairs, respectively, instead of the Watson-Crick pair C:G or mC:G. Mutations in the encoded proteins are among the consequences of such mismatches.

The

Base excision repair system is a machinery of enzymes, recognising and removing mispairs in the DNA which are ultimately replaced by the correct nucleotides. Glycosylases such as the human thymine DNA glycosylase (TDG) or uracil DNA glycosylase (UDG) are the key players in the rst step of base excision, recognising T:G or U:G mismatches and specically removing the mispaired T or U, respectively. Thymine DNA glycosylase has recently become famous also for its role in a demethylation pathway, resulting in the removal of 5mC: Enzymes such as the ten-eleven translocation (TET) methyl-cytosine dioxygenases transform 5mC through step-wise oxidation into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (1

3 ). Whereas 5-hydroxymethylcytosine (5hmC) (and mCyt) are not processed by the glycosylase enzyme, the higher-oxidised forms, 5fC and 5caC are recognised and expelled by TDG,

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Biochemistry

and ultimately replaced by unmethylated cytosine, following the base excision repair pathway (1 , 4 , 5 ). Human TDG has been crystallised in complex with DNA containing various mismatches (6 , 7 ) and damages, including G:5caC in wild-type (8 ) and mutant (9 ), with G:5hmU (5 , 9 ), an analogue (2´-uoroarabino) form of 5-formyldeoxycytidine (5fc) (10 ). From the many structures of glycosylases complexed to damaged DNA it is known that damaged, mispaired or wrong bases are ipped out of the helical DNA duplex into the enzyme's active site(11 14 ). There is still a debate how glycosylase enzymes recognise a damaged or mispaired base in an abundance of intact or properly bases (15 , 16 ). Two mechanisms are discussed. One is a passive mechanism in which the enzyme detects extra-helically exposed, already, at least partially, ipped-out bases. This mechanism implies that base pair opening up to several degrees of ipping is more likely for damaged/mispaired bases than for intact canonical ones as has been shown in some cases by computational and experimental studies (17 23 ). UDG has indeed been reported to capture a spontaneously ipped-out base (24 , 25 ). The alternative mechanism involves ipping of the base while the enzyme travels along the DNA, relying on the enzyme specically enhancing the ip-out of its target base. (15 , 25 ). Such mechanisms have been reported for the glycosylases hOGG1 and MutM (26 , 27 ), both recognising oxidatively damaged guanine. Biochemical DNA binding data show binding of TDG to C, 5mC and 5hmC to be signicantly weaker than binding to DNA with substrate bases. This has been interpreted as a discrimination step before base-ip and reactive complex formation(4 , 28 ). A particular challenge for the enzymatic specicity of TDG is the discrimination of a wrongly-placed native thymine nucleotide over a properly paired thymine in T:A. In this work, we have therefore explored the interactions between the TDG protein and DNA carrying a T:G mispair in comparison to intact DNA with a central T:A or mC:G Watson-Crick pair. To this end, we have performed molecular dynamics simulations of the complexes with the DNA in intra-helical conformation, i.e. the base is still within the DNA helix, and complexes with extra-helical conformation, i.e. the base is ipped into the active site of the protein.

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Methods Systems setup Models of the hTDG-DNA complex in the intra-helical (ipped-in) and extra-helical (ippedout) state were set up for each of the three dierent DNA models with a thymine:guanine (T:G) mispair, a methyl-cytosine:guanine (mC:G) pair, and a thymine-adenine (T:A) pair, respectively.

Starting point for the models with DNA in intra-helical state was the crystal

structure of the 2:1 TDG-DNA complex (PDB ID code 2RBA) (6 ).

We used the part in

which one of the two TDG proteins solved in the structure (chain B in the pdb structure 2RBA, containing residues 123304) is complexed to unspecic DNA, i.e. with a C:G pair in the centre and 5 more base pairs downstream and upstream, respectively, i.e. 11 base pairs in total. Cytosine has then been changed in silico to methyl-cytosine or thymine, respectively. Likewise, for the T:A model, the guanine on the complementary strand has been replaced by adenine. Models of the TDG-DNA complex in extra-helical conformation are built from the X-ray crystal structure of the human thymine DNA glycosylase bound to a substrate analogue 2'uoro-2'-deoxyuridine already ipped (but not cleaved) in the active site of the enzyme (PDB ID code 3UFJ) (7 ).

The substrate analogue was replaced in silico by thymine or methyl-

cytosine, respectively. Again for the T:A model, the guanine on the complementary strand has been replaced by adenine. Alsoin these computational models, a protein chain (chain A in the pdb structure 3UFJ, residues 120304) and a DNA strand of 11 bp length with the target base pair (T:G, mC:G, or T:A, respectively) in the centre (residue number 6), complexed to the protein, were included. The DNA sequence bound to TDG is GCTCAXGTACA for the simulations of the intrahelical and the extra-helical complex.

X marks the central thymine

or methyl-cytosine, respectively .Our simulations of the T:G model in extra-helical form have already served as starting points for subsequent quantum mechanical/molecular mechanical (QM/MM) computations of the enzymatic mechanism of glycosidic bond cleavage and are also reported there (29 ). Hydrogen atoms were placed by psfgen (VMD) (30 32 ) and the protonation state of all the

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Biochemistry

titratable amino acids was the same as in our previous simulations (29 ). In particular, His151 that has been observed to play a central role as proton shuttle in the catalytic mechanism (29 ) has been setup in its protonated form. All six models were solvated with the explicit TIP3P water model (33 ) in a cubic box of 90 Å side length such that the shortest distance between the protein-DNA complex and the edge of the simulation box is

∼ 15

Å. A total of 12 sodium counter-ions were added to

neutralise the system.

Molecular Dynamics Simulations The simulations were run with the program NAMD (34 ) using the CHARMM27 force eld (35 , 36 ) like in or earlier studies of free DNA (20 ) and the extra-helical complex of the T:G model (29 ). Non-bonding interactions were treated by means of periodic boundary conditions (switch function with a cut-o of 14-12 Å and a pair list of 16 Å). Particle-Mesh Ewald (PME (37 )) was used to calculate the periodic electrostatic interactions. The solvated (and neutralised) system were rst energy minimised (5000 steps of conjugate

−4 gradient with an energy tolerance of 10 kcal/mol) followed by a molecular dynamics (MD) simulation of 30 ps (time-step 1 fs) to heat up the system by velocity scaling. After that, a relaxation MD of 100 ps at the target temperature (time-step 1 fs) was computed for an NPT ensemble in which the pressure was maintained using the Nosé-Hoover Langevin piston with a decay period of 500 fs. Simulations of the complex with extra-helical base were performed at 295 K to be consistent with previous simulations of the complex carrying T:G (29 ).

In

that earlier work the temperature had been chosen to match that of kinetic experiments of the base-excision reaction, reported in (7 ).

Models with intra-helical conformations were

performed at to 300 K, the same temperature used for previous simulations of uncomplexed DNA (20 ). In all simulations the terminal base pairs were restraint by a harmonic potential with force constant 20 kcal/mol, centred around a 3 Å distance between the centres of mass of the respective donor/acceptor atoms of base-pair hydrogen bonds. For all models, three MD production runs of 100 ns each (time-step 2 fs) were carried out started with dierent initial velocities, except for the T:G model in intra-helical state for

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which seven independent simulations were carried out.

Free Energy Perturbation MD Simulations In order to quantify the free energy dierence of hTDG-DNA complexes, carrying a T:G mispair or a mC:G pair, we have carried out free energy perturbation (FEP) simulations In this approach (38 , 39 ) the free energy dierence

∆G between the two states can be calculated

by

∆G = GmC:G − GT :G where

kB

EmC:G − ET :G = −kB T · ln exp − kB T

is the Boltzmann constant and

T

is the temperature.

T :G

The ensemble average is

obtained from a simulation in one state (in the equation the T:G complex) and computing as well the energy of the mC:G state at every sample of T:G (and vice versa). The alchemical transition from one state

(λ = 0)

to another

(λ = 1)

has been performed by using a dual-

topology approach(40 ) for in-silico mutating one base into the other through 100 windows with

δλ = 0.01.

At each perturbation step a simulation of 2ns was carried out from which

the rst nanosecond has been considered as equilibration, thus excluded from the ensemble average. Except for the alchemical part, i.e. the dual topology, the FEP simulations were carried out under the same conditions (2 fs time steps, NPT with 300 K and 1 bar) as the unbiased MD simulations.

Three independent MD production runs, started with dierent

initial velocities, were carried out for each FEP simulation.

Analysis All molecular images were generated with the molecular visualisation program VMD (32 ). Structural analysis was performed using Curves5.3 for DNA parameters, gromacs-5.0 (41

43 )(44 ) tools and our own scripts. Hydrogen-bond occupancies were calculated as the ratio of the time when the hydrogen bond is formed to the total time of the trajectory. Two atoms are considered here to form a hydrogen bond if the acceptor-donor distance is angle is

138◦ .

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Biochemistry

For all analyses, properties were evaluated for each run individually and then averaged. Only the last 80ns were considered from the unbiased MD simulations, regarding the rst 20ns as further equilibration time.The standard deviations of the mean are used as error estimates.

Results We have performed molecular simulations of complexes of the enzyme TDG with DNA carrying a canonical mC:G pair, a T:A pair and a T:G mispair. These complexes have been simulated in a state where the central, target base is still inside the DNA helix (ipped-in state) and another, extra-helical state, with the target base ipped-out of the DNA into the enzyme's active site.

TDG-DNA complex with intra-helical (ipped-in) base DNA conformation

a)

b)

c)

d)

Figure 1:

Free energy proles of local DNA parameters a) opening, b) ip angle in free DNA

(adapted from Ref. (20 )), and c) opening, d) ip angle in the TDG-DNA complex of the T:A pair (black), T:G mispair (red), and mC:G pair (green), respectively.

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Previous work has already shown signicant dierences between the C:G or T:A pairs and the mispaired T:G in free, uncomplexed DNA. A major nding is the free energy prole of the base pair opening angle as well as the ip angle showing two local free energy minima corresponding to two states in the T:G wobble pair of free DNA (Figure 1)(20 ) For a denition of opening and ip angle see supplementary material.).

Analysis of the opening angle and

ip angle in the TDG-DNA complex shows again two local free energy minima, that is two states for the DNA at the T:G mispair. When complexed to the protein the state with an opening angle close to the free energy minimum at

∼0◦

observed for the mC:G and T:A pairs,

respectively, for T:G has a higher free energy, and hence is less favourable than the state with

◦ the larger opening angle (∼45 ). For the two base pairs, T:A and mC:G complexation by the TDG protein hardly has any eect on the opening angle. For T:A, the free energy prole is slightly widened towards larger opening angles, indicating that the T:A pair is more prone to distortion than the mC:G pair. Similarly, for the ip angle, the most probable state, i.e.

the one with the lowest free

◦ energy, for the two pairs, T:A and mC:G is a completely unipped, intra-helical state (∼40 ). In contrast, the DNA carrying the T:G mispair exhibits predominantly a partially ipped state

◦ with the same pseudo-dihedral angle value (∼70 ) as the corresponding, but less preferred ◦ state observed in the free DNA. The closed state with a ip angle of (∼50 ) is substantially reduced in probability when the DNA is bound to the TDG protein. This reverse in probability of closed and partially open states indicates that in case of the T:G mispair the interaction with the protein stabilises the distorted (partially open/partially ipped) stated.

Hydrogen bonds The distortion of the wobble, T:G mispair is further documented in the inter-base hydrogen bonds. As listed in Table 1, the mC:G pair shows proper Watson-Crick hydrogen-bonding. For the T:A pair one of the two Watson-Crick hydrogen bonds, T-N3· · · A-N1, is still very strong, whereas the other has a reduced probability compared to that observed in the simulation of free DNA (20 ). This is in agreement with the slight widening of the opening angle observed for the T:A pair. The inter-base hydrogen bonds in the T:G mispair show strong uctuations,

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Biochemistry

Figure 2: Snap shots of the MD simulations of TDG complexed to DNA with a T:A pair (black), a T:G mispair (red), and a mC:G pair (green), respectively.

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as can be seen by the relatively large errors in the computed occupancies.

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Especially the

hydrogen bond between T-N3 and G-O6 is observed in only some of the individual simulation runs. The hydrogen bonds G-N2· · ·T-O2 and G-N1· · ·T-O2 are occupied only about half of the simulation time, indicating a competition between the two. That is T-O2 is only bound to one of the two nitrogen atoms of G6, consequently exhibiting an increased opening angle. Indeed, the probability for the two hydrogen bonds to be formed simultaneously is 0.08

±

0.02. Figure 2 shows a snapshot in which the O2 atom of the mispaired thymine is situated between two hydrogen bonds to the opposing guanine base. The hydrogen bond G-N2· · ·T-O2 is an additional bond already observed in the situations of free DNA and corresponds to the distorted, partially-open state (20 ). Higher occupancy of the bond in the TDG-DNA complex is thus associated with a higher probability of the larger opening angle. It is interesting to note that no direct hydrogen bond is formed between the protein and the central base pair, T:A, mC:G, or mispair T:G, respectively. Only the complex with mC:G shows a hydrogen bond (∼

50%

occupancy) between a protein residue (Ser200) and the

phosphate group of the target nucleotide, albeit with rather large error (Table 2). Arg275, as is indicated in Figure 2, does not achieve intercalation in either complex, even in the partiallyipped conformations observed for T:G. It is, however, rather close to the phosphate backbone in all intra-helical complexes. Another residue in the proximity of the target base is Lys201. This residue can reach out of the protein into the DNA helix and eventually form a specic hydrogen bond to the O4 atom of the thymine base if that one is in a partially open conformation. Such a hydrogen bond can be speculated to help further base ip towards the active site. It must be noted that, although the T:G complex is the only one exhibiting short enough distances between Lys201 and the base to form hydrogen bonds, no such hydrogen bonds are actually observed in our simulations and there is no correlation (

Interactions of the DNA Repair Enzyme Human Thymine DNA

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