**1. Introduction**

388 Selected Topics in DNA Repair

Zhang, J. (2003). Evolution by gene duplication: an update. *Trends in Ecology & Evolution*,

(June 2003) Vol. 18, No. 6, pp.292-298, ISSN 0169-5347

520, ISSN 0829-8211

chromatin template. *Biochemistry and Cell Biology*, Vol.85, (August 2007), pp. 509–

A cells genetic information, its 'blueprint of life', is contained within its DNA. This biologically important molecule, however, can be attacked by high–energy ionizing radiation and oxidizing agents resulting in a range of possible damage. For instance, nucleobases can undergo chemical modifications or degradation such as oxidation, deamination, alkylation or be cleaved from the sugar-phosphate backbone. (De Bont & van Larebeke 2004; Friedberg et al. 2004; Hecht 1999; Kamiya et al. 1998; Labet et al. 2008a; Lindahl 1993; Lysetska et al. 2002; Neeley & Essigmann 2006; Rydberg & Lindahl 1982; Taylor 1994; Wang 2008) Similarly, the deoxyribose sugar moieties may also undergo various chemical modifications. These events can lead further to the formation of DNA– DNA or DNA–protein cross–links or DNA–strand breaks. (Kumar & Sevilla 2010; Lipfert et al. 2004) Importantly, damage to DNA can significantly affect its replication and transcription. This can ultimately result in cell apoptosis or protein mutations and pathological diseases such as cancer. (Pages & Fuchs 2002)

Experimentally, there have been numerous detailed *in vivo* and *in vitro* investigations into the processes and pathways involved in the damage of DNA. (See, for instance, Kumar & Sevilla 2010; Mishina et al. 2006, Wetmore et al. 2001) Radiolysis experiments with photometric, electrochemical and electron paramagnetic resonance detection, enzymatic inhibition and mutagenesis studies have identified a large number of reaction intermediates and rate constants for many damage and repair processes. For more in-depth reviews of experimental investigations on DNA damage processes the reader is also directed to relevant chapters in this present book. Unfortunately, however, many uncertainties and questions still remain about DNA damage and repair.

Computational chemistry provides an alternate and also complementary approach for obtaining a deeper understanding of chemical processes. This is in part because it can not only be applied to systems that are amenable to experimental investigation but also to those

Mechanisms of Mutagenic DNA Nucleobase Damages and

this chapter a dielectric constant (

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 391

Many biochemical processes occur within a polar environment, in particular an enzyme active site or aqueous medium. This can influence the properties and reactions of a biomolecule. Hence, it can be important to include such effects in the computational model. Each study described in this chapter has used a polarizable continuum (PCM)-based approach(Cammi & Tomasi 1995; Miertus et al. 1981) in the integral equation formulism (IEF).(Cances & Mennucci 1998; Cances et al. 1997; Mennucci et al. 1997) In this method the chemical system is effectively 'wrapped' in a density-fitting polar dielectric medium. Within

within a protein active site, while the standard value for water at 298 K has been used for reactions modeled in aqueous environments. The specific computational details of each

In order to determine relative free energies of reactions in which protons and electrons act as independent ions, it can be necessary to use 'fundamental values' such as the chemical potential of an electron or proton in a vacuum or aqueous solution under standard conditions. The values used in this chapter are given in **Table 1.** These have been obtained by means of a first principles quantum and statistical mechanics approach, the details of

**Quantity eV kcal mol–1** 

investigation are outlined in their respective section and in the appropriate article.

which are provided in a recent paper by Llano and Eriksson.(Llano & Eriksson 2002)

Table 1. Chemical potentials of an electron in vacuum and an electron and proton in

Ionizing radiation can potentially be absorbed by any of the three nucleotide components of DNA (i.e., phosphate, sugar or nucleobase) or its surrounding waters; also an essential part of DNA structure.(Kumar & Sevilla 2010) Primary damage of, for example, nucleobases, is caused by their direct absorption of radiation. Secondary damage (such as that described later in this chapter) can also occur, for instance, when the radiation is absorbed by the solvent, thus generating radicals or solvated electrons which then attack the nucleobase.(von

Direct absorption (primary damage) can cause the formation of a radical-cationic base via the loss of an electron, i.e., generation of an electron hole. Due to the stacking of nucleobases within DNA charge transfer (transfer of the hole) can then occur along the strand. Consequently, this enables 'primary damage related' reactions to occur distant from the site of initial damage. The potential for charge transfer due to the *π*–orbital interactions between bases was proposed as early as 1962. (Eley & Spivey 1962) Alternatively, however, it may enable charge recombination to occur further along the chain. This is because radical-anionic bases can be formed by the capture of the free electrons, where the resulting damage to the nucleobase also constitutes primary damage. Guanine has the lowest ionization potential of

aqueous reference states.(Llano & Eriksson 2002).

Sonntag 1987; von Sonntag 1996)

**2. Nucleobase oxidation via ionizing radiation** 

) value of 4.0 has been used when modeling reactions

0.0 0.0

–4.34 ± 0.02 –100.03 ± 0.5

–1.6638 ± 0.04 –38.37 ± 0.5

–11.6511 ± 0.02 –268.68 ± 0.5

ε

systems or reactions that may be too challenging or even impossible to study using such experimental techniques. Furthermore, it is nowadays possible to apply highly accurate and reliable computational methods to larger, more complete biochemical models. Thus, using such approaches one can not only reconcile theory with experiment but, for example, compare the feasibility of differing proposed reaction mechanisms or identify new pathways.

There have been numerous computational investigations on or related to DNA damage and repair. In this chapter the application of Computational Chemistry to the study of DNA damage and repair is illustrated through a review of a number of relevant computational investigations that we have performed. More specifically, we have applied high accuracy density functional theory (DFT)-based methods to the study of several important primary and secondary nucleobase damage pathways and repair mechanisms. The chapter is divided into sections, each of which focuses on select results concerning a damage and/or repair process involving either the nucleobase or phosphate components of DNA:


#### **1.1 Methods**

For the detailed study of electronic properties of biochemical systems the current computational chemistry methods of choice for investigators are those of density functional theory (DFT). The principle reasons for this is that they inherently include electron correlation effects via their basis upon the electron density of the chemical system. Such effects are often important, for example, in accurately describing bond making and breaking processes, or weakly bound systems such as reaction transition structures. Furthermore, regardless of the number of electrons in the chemical system, the electron density is only ever a function of the three Cartesian coordinates. Thus, with such methods one can use larger and more complete chemical models consisting of 150 or more atoms. In addition, they are usually highly reliable and accurate.(See, for example, Llano 2010) For biochemical systems, the currently most widely used DFT functional is B3LYP,(Becke 1993a; Becke 1993b; Lee et al. 1988) and it has been used throughout this chapter.

systems or reactions that may be too challenging or even impossible to study using such experimental techniques. Furthermore, it is nowadays possible to apply highly accurate and reliable computational methods to larger, more complete biochemical models. Thus, using such approaches one can not only reconcile theory with experiment but, for example, compare the feasibility of differing proposed reaction mechanisms or identify new

There have been numerous computational investigations on or related to DNA damage and repair. In this chapter the application of Computational Chemistry to the study of DNA damage and repair is illustrated through a review of a number of relevant computational investigations that we have performed. More specifically, we have applied high accuracy density functional theory (DFT)-based methods to the study of several important primary and secondary nucleobase damage pathways and repair mechanisms. The chapter is divided into sections, each of which focuses on select results concerning a damage and/or

2. **Nucleobase oxidation via ionizing radiation**: describes primary redox damage in nucleobases. In particular, the Gibbs Free Energies of solution-phase electron (ET), proton (PT) and proton-coupled electron (PTET) transfers for all DNA bases are examined. In addition, the potential of alkylthiols to act as repair agents of such damage

3. **8-Oxopurine formation in purine, adenine and guanine**: focuses on secondary damage of purine nucleobases. Specifically, the mechanisms of •OH attack leading to formation of 8–oxopurine derivatives are discussed as well as the Gibbs Free Energy changes of

4. **Deamination of oxidized cytosine**: examines the thermochemistry of several possible mechanisms by which oxidized cytosine, a pyrimidine nucleobase, may undergo

5. **Oxidation of serine phosphate: Implications for DNA**: investigates radicals formed

6. **Oxidative repair of alkylated nucleobases: The catalytic mechanism of AlkB**: describes key results of our studies on the enzymatic mechanism of AlkB, a member of the family of α-ketoglutarate-Fe(II)-dependent dioxygenase enzymes that catalyse the

For the detailed study of electronic properties of biochemical systems the current computational chemistry methods of choice for investigators are those of density functional theory (DFT). The principle reasons for this is that they inherently include electron correlation effects via their basis upon the electron density of the chemical system. Such effects are often important, for example, in accurately describing bond making and breaking processes, or weakly bound systems such as reaction transition structures. Furthermore, regardless of the number of electrons in the chemical system, the electron density is only ever a function of the three Cartesian coordinates. Thus, with such methods one can use larger and more complete chemical models consisting of 150 or more atoms. In addition, they are usually highly reliable and accurate.(See, for example, Llano 2010) For biochemical systems, the currently most widely used DFT functional is B3LYP,(Becke 1993a; Becke

repair process involving either the nucleobase or phosphate components of DNA:

possible associated ET, PT and PTET processes.

oxidative repair of alkylated nucleobases.

from radiation–induced damage of serine phosphate.

1993b; Lee et al. 1988) and it has been used throughout this chapter.

pathways.

is considered.

deamination.

**1.1 Methods** 

Many biochemical processes occur within a polar environment, in particular an enzyme active site or aqueous medium. This can influence the properties and reactions of a biomolecule. Hence, it can be important to include such effects in the computational model. Each study described in this chapter has used a polarizable continuum (PCM)-based approach(Cammi & Tomasi 1995; Miertus et al. 1981) in the integral equation formulism (IEF).(Cances & Mennucci 1998; Cances et al. 1997; Mennucci et al. 1997) In this method the chemical system is effectively 'wrapped' in a density-fitting polar dielectric medium. Within this chapter a dielectric constant (ε) value of 4.0 has been used when modeling reactions within a protein active site, while the standard value for water at 298 K has been used for reactions modeled in aqueous environments. The specific computational details of each investigation are outlined in their respective section and in the appropriate article.

In order to determine relative free energies of reactions in which protons and electrons act as independent ions, it can be necessary to use 'fundamental values' such as the chemical potential of an electron or proton in a vacuum or aqueous solution under standard conditions. The values used in this chapter are given in **Table 1.** These have been obtained by means of a first principles quantum and statistical mechanics approach, the details of which are provided in a recent paper by Llano and Eriksson.(Llano & Eriksson 2002)


Table 1. Chemical potentials of an electron in vacuum and an electron and proton in aqueous reference states.(Llano & Eriksson 2002).
