**2. Epigenetic alterations required for DNA repair**

 DNA is condensed in the nucleus of the cell in a highly organized and compact manner, referred to as chromatin (reviewed by Walker and Nestler [7] and Ding et al. [8]). In chromatin, the DNA is packaged with histone proteins to form nucleosomes. DNA repair proteins are recruited and interact with DNA in response to DNA damage. However, the architecture of nucleosomes and the organization of chromatin can present barriers to DNA damage recognition and repair. Epigenetic modifiers play an important role in regulating nucleosome and chromatin structure to facilitate DNA repair. Epigenetic alterations relax certain regions of chromatin to allow access to DNA repair enzymes and also condense certain regions to repress transcription in order to facilitate repair. When repair is complete, epigenetic modifications are largely returned to the state before damage occurred. These roles of epigenetic modifiers in DNA repair have been described as the "access-repair-restore" model [9].

#### **2.1 Histone acetylation**

The basic unit of chromatin, the nucleosome, is composed of 147 DNA base pairs wrapped around a histone octamer consisting of two copies of each of the following proteins: H2A, H2B, H3, and H4. The histones also have histone tail extensions, constituting up to 30% by mass of the histones (**Figure 1**). Each histone protein can undergo post translational modifications in which molecules, such as an acetyl group or one (or up to three) methyl group(s), are covalently added to (or removed from)

#### **Figure 1.**

*A nucleosome showing 4 pairs of histones (H2A, H2B, H3, and H4), each pair with the same color. The aminoterminal (N-terminal) tails of one of each pair of histones is shown, labeled with the positions of lysine amino acids (labeled "K" in the single letter code for lysine) that are subject to acetylation or methylation. The number at each lysine indicates its position counting from the amino end of the protein chain. Acetylations (indicated by Ac) and methylations (indicated by Me) are shown in some positions susceptible to these alterations. The DNA, wound around the histone core, is indicated by the dark line.* 

lysine residues of their amino terminal (N-terminal) tail. The single letter K designates lysine. If an acetyl group is added to a lysine located as the 4th amino acid from the N-terminal tail end of histone 3, this is designated H3K4Ac. These modifications not only alter the structure of the nucleosome but also change the interaction of DNA with the associated histones, thus allowing entry of DNA repair enzymes into chromatin and permitting histones to be moved, if needed, to allow for repair [2]. The epigenetic modifications, if they remain after DNA repair, also can increase or decrease the likelihood of transcription of a given gene near the site of the repaired DNA damage [10].

Acetylation of histone lysines promotes chromatin relaxation to facilitate DNA repair [2]. It is also generally associated with a permissive transcriptional state. By negating the positive charge associated with the lysine residues on histone tails, acetylation promotes an "open" chromatin state.

#### **2.2 Histone methylation**

Histone lysine methylation is associated with either activation or repression of gene expression depending on which residues are methylated and whether one, two or three methyl groups are added at that position [11]. For instance enrichment of H3K4Me1 or H3K4Me3 at specific gene regions is correlated positively with increased transcription levels, whereas enrichment of H3K9Me2, H3K9Me3 or H3K27Me3 is negatively correlated with transcription. In response to DNA double-strand breaks, histone methyl transferases are recruited to sites of damage where they catalyze trimethylation of H3K9 and H3K27, thereby repressing transcription in order to facilitate DNA repair [12].

#### **2.3 DNA methylation**

An important epigenetic regulator in addition to histone modification is DNA methylation. DNA methylation often occurs with the addition of a methyl group to the DNA sequence cytosine-phosphate-guanine (CpG) at the C5 position (5mC). DNA methylation at gene promoters is generally associated with repression, while methylation within genes has been associated with active transcription [7]. DNA methyl transferase 1 (DNMT1) binds to sites of oxidative damage formed in GC-rich regions of the genome and promotes formation and recruitment of a large epigenetic silencing complex. Localization of these epigenetic modifiers to sites of oxidative damage in promoter CpG islands results in increased DNA methylation.

#### **2.4 Noncoding RNA**

Non-coding RNAs provide an additional type of epigenetic regulation. As one example, microRNAs are short sequences of RNA (about 22 bases) that exert a repressive role on gene expression by binding a target sequence on specific mRNAs and blocking translation or inducing degradation. The typical microRNA has about 400 specific target mRNAs. In one report, specific microRNAs collaborated with histone deacetylases and cooperatively regulated several relevant target genes [13].

#### **2.5 Epigenetic alterations allow DNA repair**

**Figure 2** illustrates some of the actions of epigenetic alterations. Histone acetyltransferases add acetyl groups to histone tails to open chromatin structure to make DNA damages accessible to repair enzymes. If the acetyl groups remain after DNA repair, this allows genes in the area of DNA repair to be switched on. Histone deacetylases remove acetyl groups from histone tails to complete DNA repair and return chromatin to its condensed state existing before DNA repair. Improper actions of histone deacetylases can inappropriately switch genes off.

*The Role of DNA Repair and the Epigenetic Markers Left after Repair in Neurologic Functions… DOI: http://dx.doi.org/10.5772/intechopen.83477* 

#### **Figure 2.**

*A gene located in chromatin, with its DNA wrapped around histones. Open chromatin (top image) has been relaxed by acetylation of histone tails. Condensed chromatin (lower image) has been tightened by removal of acetyl groups and addition of methylation of histone tails. Symbols include histone acetyltransferase (HAT), histone deacetylase (HDAC), and histone methyltransferase (HMT). SWI/SNF (not an epigenetic alteration) is a nucleosome-remodeling complex that stimulates gene expression if the epigenetic factors (methylation on cytosines in DNA; acetylations and methylations of lysines in histone tails) allow transcription [14].* 

In this figure, histone methyltransferase (HMT) is shown as switching a gene off. However, some histone methylations serve to activate genes [11].

DNA gene promoters without methylated cytosines are indicated (in **Figure 2**) as allowing transcription (upper image), and DNA gene promoters with methylated cytosines (in CpG sites) are indicated as impeding transcription. The green vertical ovals represent transcription factors. They are not epigenetic factors, but also regulate gene transcription.

#### **3. Epigenetic remnants (scars) after DNA repair**

Dabin et al. [15], in an extensive review, noted that after repair of various types of DNA damages there are a number of types of epigenetic alterations that could potentially remain as scars. These epigenetic alterations include (1) changes in DNA methylation, (2) incorporation into nucleosomes of new histones with a pattern of acetylations or methylations that differ from that in the histones originally present before DNA damage, (3) incorporation into nucleosomes of histone variants such as histone H2AZ, (4) altered acetylation or methylation of histone tails, and even (5) altered histone density at repair sites. Several illustrative reports showing such scars after DNA repair are described below.

#### **3.1 Homologous recombinational repair (HRR) of double-strand breaks leaves epigenetic alterations**

Homologous recombinational repair (HRR) modified the DNA methylation pattern of a repaired DNA double-strand break in a green fluorescent protein (GFP) gene inserted into the HeLa cell genome [16]. In different subclones isolated after HRR repair events, the repair created either more highly methylated or less highly methylated cytosines in the GFP gene DNA. HRR also altered local histone H3 methylation,

#### *DNA Repair - An Update*

forming H3K9Me2 or H3K9Me3 at repair locations. However, H3K9Me2/3 was selectively retained after HRR only in recombined genes with increased DNA methylation.

During a 2-week period after repair, some transcription-associated demethylation of the repaired DNA was promoted by base excision repair enzymes [16]. Subsequently, the repaired genes displayed stable but diverse methylation profiles. These profiles governed the levels of expression in each clone. These epigenetic alterations (scars) were stable over time and were recovered with the same frequency after 3 years of continuous culture.

#### **3.2 Double-strand break repair by non-homologous end joining leaves epigenetic alterations**

DNA repair by non-homologous end joining induces alterations in DNA cytosine methylation and these alterations are a source of permanent epigenetic changes [17]. In a HeLa cell line containing a green fluorescent protein (GFP) based reporter gene, a double-strand break in the gene followed by non-homologous end joining repair created two populations of cells, those with increased DNA methylation in the GFP gene (identified by a dim green color) and those with decreased DNA methylation in the GFP gene (identified by a bright green color). The degree of methylation for each population changed somewhat over the subsequent 4 days, but then remained stable for 24 days. Even though the HeLa cells were undergoing replication, the epigenetic changes produced stable high expressing or low expressing clones.

#### **3.3 Base excision repair is associated with epigenetic alterations**

The major forms of oxidative DNA damage are non-bulky lesions such as 8-oxo-2′-deoxyguanosine and thymine glycol that are repaired predominantly by base excision repair. After oxidative DNA damage was increased in HCT116 cells in culture, histone alterations were found in genes with CpG island-containing gene promoters and these histone alterations caused decreased transcription [18]. The histone alterations introduced by increased oxidative damage included reduction in H3K4Me3 and H4K16Ac and an increase in H3K27Me3. DNA methylation was also increased, but primarily in promoters of genes which normally have low basal expression [18].

#### **3.4 Histone replacements during DNA repair**

For many types of DNA damage, histones must be removed and replaced during the repair of the damaged DNA [19]. Disruption of nucleosomes in human cells after introduction of double-strand breaks or UV damage occurs with a drop in histone H2B levels and a selective loss of histones H2A and H2B, but not of H3 or H4 at the site of the damage [19]. After DNA repair, new histones (in addition to some pre-existing histones) are deposited at the site of repair. The new histones lack the histone post-translational modifications that existed before the repair. The presence of the differently modified new histones can specifically mark the domain as a site of repair, and remain as a scar [19]. The failure to recycle all of the pre-existing histone marks results in alterations in gene expression [15].

### **4. Addiction**

One of the principal features of addiction is its persistence. The persistent behavioral changes appear to be due to long-lasting changes, resulting from epigenetic alterations affecting gene expression, within particular regions of the brain [20].

*The Role of DNA Repair and the Epigenetic Markers Left after Repair in Neurologic Functions… DOI: http://dx.doi.org/10.5772/intechopen.83477* 

## **4.1 Alcohol**

 Alcohol can be addictive. About 7% of the US population are alcoholics, with alcohol use disorder [21]. Many negative physiologic consequences of alcoholism are reversible during abstinence. Long-term chronic alcoholics suffer a variety of cognitive deficiencies [22]. Multiyear abstinence resolves many neurocognitive deficits. One exception is lingering deficits in spatial processing [23]. In addition, some frequent long-term consequences are not reversible during abstinence. Alcohol craving (compulsive need to consume alcohol) is usually present longterm among alcoholics [24]. Among 461 individuals who sought help for alcohol problems, follow-up was provided for up to 16 years [25]. By 16 years, 54% of those who tried to remain abstinent without professional help had relapsed, and 39% of those who tried to remain abstinent with help (such as Alcoholics Anonymous) had relapsed.

 Long-term, stable consequences of chronic alcohol abuse are thought to be due to stable changes of gene expression resulting from epigenetic alterations within particular regions of the brain [26–28]. For example, in rats exposed to alcohol for up to 5 days, there was an increase in histone 3 lysine 9 acetylation in the pronociceptin promoter in the brain amygdala complex. This acetylation is an activating mark for pronociceptin. The nociceptin/nociceptin opioid receptor system is involved in the reinforcing or conditioning effects of alcohol [29].

#### **4.2 Cigarette smoking**

 Cigarette smokers (about 21% of the US population in 2013) [30] are usually addicted to nicotine [31]. This is a strong addiction. The proportion of smokers who reported having seriously tried to quit and who managed to quit for 6 months or more was less than 10% [32].

 After 7 days of nicotine treatment of mice, the post-translational modifications consisting of acetylation of both histone H3 and histone H4 was increased at the *FosB* promoter in the nucleus accumbens of the brain, causing a 61% increase in FosB expression [33]. This also increases expression of the splice variant *Delta FosB*. In the nucleus accumbens of the brain, Delta FosB functions as a "sustained molecular switch" and "master control protein" in the development of an addiction [34, 35]. Similarly, after 15 days of nicotine treatment of rats, the post-translational modification consisting of threefold increased acetylation of histone H4 occurs at the promoter of the dopamine D1 receptor gene in the prefrontal cortex of the rats. This caused increased dopamine release in the prefrontal cortex reward-related brain region, and such increased dopamine release is recognized as an important factor for addiction [36].

#### **4.3 Cocaine**

Cocaine addiction occurs in about 0.5% of the US population. In humans treated for cocaine addiction, the relapse rate after 5 years was 25% [37]. Repeated cocaine administration in mice induces post-translational modifications including hyperacetylation of histone 3 (H3) or histone 4 (H4) at 1696 genes in one brain reward region, the nucleus accumbens, and deacetylation at 206 genes [7, 38]. At least 45 genes, shown in previous studies to be upregulated in the brain nucleus accumbens of mice after chronic cocaine exposure, were found to be associated with post-translational hyperacetylation of histone H3 or histone H4. Many of these individual genes are directly related to aspects of addiction associated with cocaine exposure [38].

#### **4.4 Addictive substances can cause DNA damage**

In rodent models, many addictive substances cause DNA damage in the brain. For example, alcohol, through its metabolic product acetaldehyde, induces doublestrand breaks in DNA in the mouse brain [39].

 Nicotine from cigarette smoke also very likely causes DNA damage in the brain. Nicotine reaches the brain 10–20 seconds after a puff of smoke. The level of nicotine in the brain is 75–80% as high as in the blood or the liver [40]. E-cigarette smoke is composed primarily of nicotine vapors. Nicotine from E-cigarettes, applied to mice (with the dose and duration equivalent in human terms to light E-cigarette smoking for 10 years), caused DNA damages including mutagenic O6 -methyl-deoxyguanosines and γ-hydroxy-1,N2-propano-deoxyguanosines in the lung, bladder, and heart [41]. These same damages are likely to occur as well in neurons upon exposure to nicotine.

Cocaine [42] and methamphetamine [43, 44] each also cause DNA damage in the brain.

After repair at the sites of DNA damages caused by drugs of addiction, the epigenome may not return entirely to their pre-damage states. Some of the methylations of DNA and/or the acetylations or methylations of histones at the sites of DNA repair may remain and thus become epigenetic scars on chromatin [15]. Such epigenetic scars likely contribute to the persistent epigenetic alterations found in addiction.
