Computational Studies of the Mechanical Stability for Single-Strand

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Computational Studies of the Mechanical Stability for Single-Strand Break DNA Pawel Krupa, David J. Wales, and Adam K. Sieradzan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b05417 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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The Journal of Physical Chemistry

1

Computational Studies of the Mechanical Stability for Single-Strand Break DNA

Pawel Krupa,1,2,∗ David J. Wales3 and Adam K. Sieradzan2

1

Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46, PL-02668 Warsaw,

Poland, 2 Faculty of Chemistry, University of Gda´ nsk, Wita Stwosza 63, 80-308 Gda´ nsk, Poland, 3

Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW, U.K.

∗

Corresponding author; phone: +48 22 116 35 45; e-mail: [email protected] ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract The stability of DNA is crucial for the existence of most living organisms. Even a single DNA break can lead to serious problems, and even cell death. In this work the position-specificity of single strand breaks (SSB) and the stability of short DNA fragments of various lengths and sequence repetitions (d(AT )30 , d(AT GC)15 , d(GC)30 , d(T T AGG)12 , d(T T AGGG)10 , and d(T T T AGGG)9 with SSBs and d(GC) with 2-60 repetitions without SSBs) were examined, by performing a series of steered molecular dynamics simulations using the coarse-grained NARES2P force field. Our results show that the stability of DNA with a SSB strongly depends on the position of the break, and that the minimum length of DNA required for stability is sequence dependent. d(GC)30 with SSB in position x was found to be less resistant to stretching than d(GC)x without SSB, where x is the number of d(GC) repetitions. DNA sequences with longer repeated fragments (such as telomeres) exhibit greater stability in the presence of breaks positioned at the beginning of the chain, which could constitute a cellular defence mechanism against DNA damage.

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Introduction

The study of basic mechanisms involved in cell function is currently one of the most active areas in molecular science. DNA is a principal cellular component: it is responsible for carrying the genetic information of all proteins, and determines the frequency of transcription or replication. However, DNA is very prone to damage, which can lead to cancer 1 or neurodegenerative disease. 2 Moreover, DNA damage is also associated with autoimmune conditions 3 and diabetes mellitus type 2, 4 while aberrations of telomeres can lead to cancer. 5 Radiation, 6 reactive oxygen species, 7 and chemical reactants 8 are among the agents that damage DNA. In adults •

over 1 kg of superoxide radical O2− is produced annually. 9 There are several different examples of DNA damage: single strand breaks (SSBs), 10 the appearance of 8-hydroxydeoxyguanosine residues, 7 creation of polycyclic aromatic hydrocarbon adducts, 11 and breaks in double strand helices (DSB). 12 Among the various examples of DNA damage, SSBs are the most common, arising at a frequency of tens of thousands per cell per day. 13 Unless quickly repaired the most likely consequence of SSBs is collapse or blockage of replication forks during the S phase of the cell cycle, leading to DSB, and finally to overall DNA instability. 14,15 In most cases, SSBs are rapidly reversed and do not lead to DSB, 16,17 but otherwise they can lead to serious damage to neuronal cells and even to heart failure. 18 The first attempts to study the influence of SSBs on the stability and structure of the DNA started in the 1990s, when only 1 ns simulations could be performed for very small systems. 19 With advances in computational power and tools, longer simulations for larger systems with higher accuracy became possible. However, with all-atom methods, we are still limited to relatively small systems. A popular size for DNA studies is often the dodecamer on a submicrosecond timescale. 20 Recently, larger systems, e.g. 31 bp DNA, have become accessible, however, with run lengths still limited to 100 ns and with a low number of replicas. 21 For pulling velocities of 1-12 cm/s 21 significantly higher force peak values were observed than in experiment. In the present contribution we consider the mechanical stability of undamaged DNA of ACS Paragon Plus Environment


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4 various lengths compared to SSB-DNA. We apply steered molecular dynamics with the coarsegrained force field NARES-2P to study the effect of the SSB position on the mechanical resistance of damaged DNA using slow pulling speeds of 0.4 mm/s and 0.02 mm/s with at least 64 trajectories per system. We study different sequences: three non-telomeric and three telomeric, which differ in their mechanical properties. 22 We determine if the break position has a similar effect on mechanical stability to shortening of the DNA. The advantages of using an efficient coarse-grained force field facilitate: (i) using relatively low pulling speeds of 0.4 mm/s and 0.02 mm/s, (ii) performing 64 to 120 trajectories for each system, providing 7,080 trajectories in total, (iii) using larger systems, which can exhibit stability during simulations, each consisting of at least 60 bp. Our results show that stability of the single-break DNA is both position- and sequence-dependent, and that shorter DNA fragments without defects are more stable than long DNA with SSB in one of the chains in an analogous position.

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Methods

The influence of the length of broken fragments on the stability of DNA during pulling was determined using canonical SMD simulations for the NARES-2P force field, implemented in recent work. 22 Each simulation consisted of 64 trajectories, each of approximately 150 µs (30,000,000 steps, each of 4.89 fs, taking into account the approximate 3 to 4 order-of-magnitude speed-up associated with the NARES-2P force field), which were continued to full dissociation of the chains. A pulling speed of approximately 0.4 mm/s and a force constant of 2 kcal/(mol ˚ A2 ) for the harmonic spring were used in all simulations. Additionally, a series of runs with a 0.02 mm/s pulling speed (2.45 ms, 500,000,000 steps) were run to analyse the influence of the pulling speed on the results. In previous work 22 we adjusted pulling speed by trial and error and found that in some cases even with a pulling speed of 0.4 mm/s system may not have enough time to equilibrate during simulations, however, most of the regrabbing phenomenon could still be observed. In all simulations, restraints (anchors for pulling) were placed on the second residue from the 5′ position of each chain, so pulling was performed parallel to the long ACS Paragon Plus Environment

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5 axis of the DNA. NARES-2P 23,24 is a coarse-grained model for nucleic acids derived analogously to the physics-based coarse-grained model for proteins UNited RESidue (UNRES), which has been developed for over 20 years and used mostly to predict protein structure 25 and dynamics. 26 In NARES-2P only two interaction sites per nucleic acid are employed: a united sugar-base and a united phosphate group. In both force fields the energy function is derived from a Restricted Free Energy (RFE) or Potential of Mean Force (PMF) for all-atom systems, including solvent and counter-ions. All energy terms in NARES-2P are analogous to UNRES, including the temperature dependence (f2 (T )), 23 and the effective energy function is summarised in eq. (1). N ARES−2P GB Unucl = wBB

XX

+ wP P

XX

i

i 120pN 23 8 12 10 8 9

Table 2: Fitting of the obtained distance and force values from the simulations to eq. 1. d(GC)30 d(GC)30 − slow d(GC)30 − weak d(AT GC)15 d(AT )30 d(T T T AGGG)9 d(T T AGGG)10 d(T T AGGG)10 − slow d(T T AGG)12 d(GC)x d(GC)x − slow

∆Gbp /δ 801.81 703.13 843.06 556.93 499.54 524.50 537.33 516.36 516.38 995.48 606.20

error 10.39 10.58 13.39 6.87 7.75 7.12 11.07 14.76 5.54 24.42 19.02

∆Gτobs /δ 6395.8 7484.3 6889.7 3176.6 7167.5 3095.1 2660.0 3851.2 3090.2 8557.9 7130.6

error 164.8 220.9 251.5 93.7 162.4 97.1 135.9 234.5 82.5 582.1 446.3


∆Gτobs /∆Gbp 7.977 10.644 8.172 5.704 14.348 5.901 4.950 7.458 5.984 8.597 11.763

error 0.178 0.270 0.269 0.153 0.237 0.167 0.232 0.401 0.146 0.545 0.637


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Figure 1

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Figure 2

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Figure 3


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Figure 5


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Peak value [nN]

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d(GC)X d(GC)X−slow 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60

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Figure 6 ACS Paragon Plus Environment

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TOC Graphic

Stability of the DNA with SSBs Peak value [nN]

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Computational Studies of the Mechanical Stability for Single-Strand

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