Chronic Obstructive Pulmonary Disease: From Injury to Genomic Stability

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is the fourth cause of death in the world and it is currently presenting a major global public health challenge, causing premature death from pathophysiological complications and rising economic and social burdens. COPD develops from a combination of factors following exposure to pollutants and cigarette smoke, presenting a combination of both emphysema and chronic obstructive bronchitis, which causes lung airflow limitations that are not fully reversible by bronchodilators. Oxidative stress plays a key role in the maintenance and amplification of inflammation in tissue injury, and also induces DNA damages. Once the DNA molecule is damaged, enzymatic mechanisms act in order to repair the DNA molecule. These mechanisms are specific to repair of oxidative damages, such as nitrogen base modifications, or larger DNA damages, such as double-strand breaks. In addition, there is an enzymatic mechanism for the control of telomere length. All these mechanisms contribute to cell viability and homeostasis. Thus, therapies based on modulation of DNA repair and genomic stability could be effective in improving repair and recovery of lung tissue in patients with COPD.

KEYWORDS:

• DNA repair
• genomic stability
• inflammation
• oxidative stress

Introduction

According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) (1), chronic obstructive pulmonary disease (COPD) is the fourth cause of death in the world and it is currently a major challenge to public health. COPD causes chronic morbidity and mortality, giving rise to premature death from pathophysiological complications, rising economic and social burdens (2). There is expectation that deaths will increase in the next decades due to continued exposure to COPD risk factors and the aging population.

Chronic obstructive pulmonary disease is estimated to affect about 7 million people only in Brazil (3). About 12% of patients are diagnosed and only 18% of those receive some treatment, including palliatives. COPD is the fifth cause of death and hospitalization of about 290,000 patients annually, and generates excessive costs for the health care system (3). It is estimated that 24 million people in the United States were affected by COPD in 2013 (4).

The factors that trigger COPD have individual characteristics, such as epigenetic alterations, polymorphism or mutation in specific genes, genetic susceptibility, and increase of oxidative stress, which contributes to the inflammatory response (5).

When an inflammatory response initiates the development of COPD, there is an increasing generation of free radicals, establishing oxidative stress, which ultimately damages DNA and other molecules in lung cells (6). Indeed, in addition to direct and indirect DNA damages, free radicals could act as second messengers in specific cell signaling pathways, altering gene expression (7).

Despite a number of different DNA damages caused by free radical attacks, genomic integrity is achieved by a set of enzymatic DNA repair mechanisms, which recognize and repair damages in the DNA molecule (8,9). The main DNA repair mechanisms, to fix DNA damages caused by free radicals, are the base excision repair and nucleotide excision repair.

Chronic obstructive pulmonary disease: Pathophysiology

Chronic obstructive pulmonary disease affects 10–14% of world population, and in 2005 three million people who had COPD died. Estimates show that the total number of deaths in the next 10 years will increase above 30% (1,10,11). Until 2030, COPD will be the fourth cause of death worldwide (11). The increase in mortality will be due to the expanding epidemic of smoking, reduced mortality from other common causes of death (e.g. ischemic heart disease and infectious diseases), and aging of the world population (1)

According to GOLD (1), the morbidity and mortality vary across countries. Tobacco smoking is strongly related to developed countries whereas on developing countries the COPD would begin from the air pollution mainly.

Chronic^{Figure 1} obstructive pulmonary disease is a common, preventable, and treatable disease (1). Although treatment is available, various host factors could influence the pathogenesis, such as immunologic defects; genetic susceptibility; infections and environmental factors, the latter being assigned as a major risk factor; and smoking, due to a number of harmful chemical compounds present in tobacco (12).

Chronic obstructive pulmonary disease is characterized by persistent airflow limitation that is usually progressive and associated with increased chronic inflammation in airways and lungs in response to harmful particulate matter and gases (1). Also, COPD presents inflammation; fibrosis and deposition of mucus in airways, causing chronic bronchitis (10,13); and alveolar destruction in pulmonary parenchyma, resulting in the loss of elastic recoil capacity which characterizes emphysema (10). Destruction of parenchyma is related to loss of lung tissue elasticity, which occurs as a result of the destruction of supporting structures that feed the alveoli, with the small airways tending to collapse during exhalation, thus reducing airflow, increasing air retention in lungs, and causing reduction of pulmonary capacity (14).

Emphysema is an important component of COPD and it is defined as the presence of permanent increase in distal air space to terminal bronchioles followed by destruction of their walls, without clear fibrosis signals (15).

The inflammatory responses by activating the innate and adaptive immune system have been lightened by chronic exposure to the initiating agent of COPD (16). For example, cessation of smoking cigarettes increases the inflammatory process (17).

The innate response will begin with the change in the barrier between the lung tissue and airspace, due to exposure to risk factors such as smoking (14), recruiting neutrophils, macrophages, T-lymphocytes and natural killer cells to alveolous (14) (Figure 1). The adaptive immune response is started when particles undergo phagocytosis and presented as antigens by dendritic cells to T- and B-lymphocytes (18). Several cytokines and chemokines, such as interleukins (IL-1β, IL, 6 e IL-8), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), C-reactive protein (CRP), and matrix-metalloproteinases (MMP-6, MMP-9 and MMP-12), are secreted by inflammatory and epithelial cells, thereby perpetuating inflammation (19,20). These inflammatory mediators, besides perpetuating inflammation, give rise to tissue injury, as well as a range of systemic effects (20). The inflammatory response directly causes airway remodeling and narrowing of lungs in COPD patients. Airway space narrowing is caused by peribronchial fibrosis, build-up of scar tissue from damage of airway space, and over-multiplication of epithelial cells, lining the airway space. Also, mucociliary dysfunction by increase in the number of mucosal glands due to inflammation contributes to excess mucus production in airway space, which blocks the air flow (21).

Figure 1. Schematic representation for the development of COPD until the increase of free radicals and their consequences to DNA molecule. COPD, chronic obstructive pulmonary disease; TNF-α, tumor necrosis alpha factor; INF-γ, interferon gamma; CRP, C-reactive protein; MMPs, matrix-metalloproteinases.

In addition to all these changes, there is imbalance between proteinases and antiproteinases, leading to tissue changes in COPD (22). The increase in the number of neutrophils and macrophages releasing enzymes, such as cathepsin, elastase, proteinase 3, and matrix metalloproteinases 9 and 12, induces proteolysis of extracellular matrix components, resulting in the disruption of alveoli, causing emphysema (23). It was shown that increase in MMP-9 results in increased production of its inhibitor TIMP-1- to repair the damaged tissue, but the increase in inhibitor could contribute to increased deposition of extracellular matrix, and consequently tissue fibrosis (24).

Not only can the imbalance between proteinases and antiproteinases cause tissue destruction, but the oxidative stress could also cause redox imbalances (increase in destructive free radicals levels, as reactive oxygen species—ROS), reducing antioxidant protection pathways, thus perpetuating the inflammatory process (25). Oxidative stress plays a key role in tissue injury and maintenance, and amplification of inflammation. Although the lung epithelium is an important source of antioxidants, individuals with emphysema present imbalance between antioxidant and oxidant agents, with predominance of oxidizing agents, and this imbalance could cause damages to the DNA molecule, in proteins and lipids, as it has been suggested (26). Some clinical studies are listed in Table 1 correlating COPD with DNA damage or DNA alteration.

Table 1. Summary of clinical studies on the relationship between COPD and DNA damage.

				Clinical studies/
First author	Title	Reference	n	type of intervention	Lifestyle	Conclusion
Wu 2007 (126)	Vitamin E and vitamin C supplementation in patients with chronic obstructive pulmonary disease.	Int J Vitam Nutr Res;77(4):272–279.	35	Vitamin E or Vitamin C supplementation.	Patients with COPD.	Vitamin E or C supplementation may improve DNA's resistance against oxidative stress.
Leidinger P. 2011 (127)	Specific peripheral miRNA profiles for distinguishing lung cancer from COPD.	Lung Cancer;74:41–47.	71	Through the identification of microRNAs, differentiate COPD from lung cancer.	28 lung cancer patients;24 COPD patients.19 healthy patients.	MicroRNA expression should distinguish lung cancer patients from COPD patients, assigning microRNAs a biomarker.
Silva ALG. 2016 (128)	DNA damage and repair capacity in lymphocytes of chronic obstructive pulmonary disease patients during physical exercise with oxygen supplementation.	Multidiscip Respir Me;11: 43.	25	Exercises with/without O2 supplementation.	COPD patients.	COPD patients who performed exercises with oxygen supplementation had DNA damage and increased DNA repair.
Avriel A. 2016 (129)	Prognostic utility of admission cell-free DNA levels in patients with chronic obstructive pulmonary disease exacerbations.	Int J Chron Obstruct Pulmon Dis.11:3153–3161.	62	Assessment of cfDNA levels in patients admitted to hospital for a COPD exacerbation.	Patients with COPD exacerbations.	There is a possibility of using plasma cfDNA as a new technique to identify patients with poor prognostic COPD.
Carpagnano GE. 2016 (130)	Study of mitochondrial DNA alteration in the exhaled breath condensate of patients affected by obstructive lung diseases.	J. Breath Res.10:026005	62	Evaluation of mitochondrial DNA as a possible marker of oxidative stress.	13 COPD patients;14 asthmatics;23 COPD and asthma-COPD overlap syndrome;12 healthy subjects.	Positive correlation with concentration of markers in blood, and increase of exhaled marker of oxidative stress in obstructive lung diseases.
Xie JG. 2004 (131)	Smoking, the level of DNA adducts and chronic obstructive pulmonary disease.	Zhonghua Jie He He Hu Xi Za Zhi;27(7):469–473.	88	Correlate DNA adducts with smoking in the onset of COPD.	88 patients COPD;68 healthy patients smokers and non-smokers;87 healthy smoking control;	They observed an association between DNA adduct levels with smoking, so that DNA adducts can result in cell damage and contribute to COPD pathogenesis.
Anes AB. 2014 (132)	Increased oxidative stress and altered levels of nitric oxide and peroxynitrite in Tunisian patients with chronic obstructive pulmonary disease: correlation with disease severity and airflow obstruction.	Biol Trace Elem Res;161(1):20–30	382	Comparing nitric oxide and peroxynitrite levels in COPD patients and assessing the impact on disease stage and airflow obstruction.	153 COPD patients;229 Healthy patients.	DNA damage was identified, along with the presence of systemic oxidative stress.

Oxidative stress in inflammation: DNA damage

Oxidative stress acts on a variety of inflammatory mediators, and relationship between oxidative stress and inflammation is in the overproduction of ROS, which represents an important component in the development of several chronic diseases (27). Biomarkers of oxidative stress (e.g., hydrogen peroxide, 8-isoprostane) are increased in exhaled breath condensate, sputum, and in systemic circulation of COPD patients (1).

Inflammation as a natural defense mechanism acts against a variety of harmful factors, as infections, exposure to allergenic, chronic diseases, and smoking. COPD and other chronic diseases are related to increased production of free radicals, which could cause oxidation of proteins, lipids, and nucleic acids, and these are recognized as an inflammatory signal (28,29). These suggest that inflammation worsens clinical aspects of some diseases due to increased oxidative stress, by releasing macrophage-oxidized proteins, such as a peroxiredoxin 2 (PRDX2), which trigger TNF-α production in the extracellular environment, indicating that these mechanisms depend on redox in oxidative cascades and could induce inflammation (28).

Exacerbated acute inflammation also worsens prognosis of patients with COPD, being associated with increased morbidity and mortality (30). The exacerbation is associated with systemic inflammation and physiological changes caused mostly by viral and bacterial infections (31). These exacerbations include breathlessness, cough, increased mucus production, and extreme fatigue, for example, resulting in decline of lung function (32). Also, the infectious conditions cause lung injury and development of sepsis, which manifest as acute respiratory distress syndrome (ARDS), increase in free radicals in lung cells (6,33). COPD exacerbations are considered as aggravations of underlying chronic inflammation in the airways. However, data on inflammatory responses, oxidative stress, and consequently their association with DNA damage in lungs in case of COPD exacerbation are scarce (34). Studies have shown that there is increase in activated neutrophils in airways of COPD patients indicating that they are attracted by cytokines, such as IL-8 and TNF; increased cytokine levels during exacerbations in COPD patients has been reported (35,36), and the frequency of exacerbations has been associated with the severity of COPD (37).

Cell damage caused by oxidative stress alters mitochondrial DNA, protein structure (38), as well cell membranes, such as the plasma lipid peroxidation, in the endoplasmic reticulum and Golgi apparatus (39).

Free radicals are highly reactive atoms or molecules, which contain unpaired electrons in their last electronic layer, and they are produced as byproducts during normal metabolism and exposure to sunlight, ultraviolet light, ionizing radiation and toxic chemicals, such as cigarette smoke (40). Among these, there are the reactive oxygen species (ROS), which are generated in the cells by both oxidizing and reducing agents (41). Hydroxyl radical (OH˙), superoxide (O₂˙), hydrogen peroxide (H₂O₂) ozone (O₃), and singlet oxygen (¹O₂) are examples of ROS (41). Besides ROS, reactive nitrogen species (RNS) are produced in vivo from arginine by nitric oxide synthase (NOS) and play important roles in vascular relaxation, biological defense, neurotransmission modulation, and immune system (41). Nitric oxide (NO) and peroxynitrite (ONOO⁻) are examples of RNS (41).

Free radicals can act as second messengers in specific cell signaling pathways, in immune function, and signal transductions, thereby affecting cellular homeostasis (42). Some studies have shown that activation of NF-kB (nuclear kappa factor B) and AP-1 (activator protein 1) promotes cell proliferation and survival (43,44). Furthermore, increase in ROS and RNS is related to stabilization of HIF1α (hypoxia-induced factor 1 alpha), which leads to transcription of a number of genes involved in cell metabolism, survival, and death; angiogenesis; invasion; and metastasis (45). Also, ROS regulates serine/threonine AMP-activated protein kinase (AMPK), which contributes to energy metabolism (46).

On the other hand, these free radicals can react with the DNA molecule, mainly causing changes in nitrogenous bases producing oxidized bases or crosslinking between two adjacent bases (47). However, after damage, the DNA molecule can be repaired by enzymatic mechanisms (48), being crucial for maintenance of genome integrity.

In fact, pathogenesis of COPD leads to increased oxidative stress, resulting in damages in lung cells, mucus hypersecretion, inactivation of antiprotease, and exacerbation of inflammation (49). Studies indicate that DNA repair inefficiency is common in COPD patients and correlates with susceptibility to disease development and progression (50), and that in a hypoxic response condition, DNA damage is prominent in lung cells of COPD patients (51). Another study suggests that increased damage and recurrent repair, linked to the condition of hypoxia, could deplete the regenerative capacity of satellite cells in COPD patients, causing muscle atrophy (52). Also, there is an increase in DNA damage under oxygen supplementation conditions, but the response of DNA repair mechanisms is more effective (53). Fetal lung cells exposed to cigarette smoke present breaks in DNA chain, as well as initiation of DNA repair (54). A study carried out with 100 healthy subjects showed that DNA fragmentation is significantly increased in smokers, mainly in women (55). These results were confirmed by Mercken et al. (56), where an immediate increase in DNA damage was observed in patients with COPD who underwent physical exercise tests. Moreover, it has been suggested that oxidative stress may extend beyond the lung to generate systemic manifestations of COPD (57,58) and oxidative stress and DNA damage are increased in peripheral blood cells from COPD patients (59). In addition, there are evidences relating oxidative stress and pro-inflammatory mediators to epigenetic changes, such as nuclear histone acetylation/deacetylation, restricting transcription factors to DNA, and thereby altering the rate of expression of pro-inflammatory genes (60). However, there are no data indicating restriction of transcription factors that alter gene expression related to DNA repair in COPD patients. Table 2 lists studies on COPD and DNA changes, damage, and repair.

Table 2. Summary of studies on the relationship between COPD, DNA damage and DNA repair mechanisms.

			Alteration/	DNA repair
First author	Title	Reference	damage	mechanisms	Model
Amsellen V. 2011 (109)	Telomere dysfunction causes sustained inflammation in chronic obstructive pulmonary disease.	Am J Respir Crit Care Med;184(12):1358–1366	Telomere dysfunction	Unidentified	Human
Alder JK. 2011 (112)	Telomere length is a determinant of emphysema susceptibility.	Am J Respir Crit Care Med;184(8):904–912.	Telomere dysfunction	Unidentified	Mice
Tzortzaki EG. 2012 (5)	Oxidative DNA damage and somatic mutations: A link to the molecular pathogenesis of chronic inflammatory airway diseases.	Chest; 141(5):1243–1250.	Mutations/oxidative	BER	Human
da Silva AL. 2013 (133)	Evaluation of DNA damage in COPD patients and its correlation with polymorphisms in repair genes.	BMC Med Genet;20:93	Polymorphisms/double strand break	BER, HR, NHEJ	Human
Valavanidis A. 2013 (6)	Pulmonary oxidative stress, inflammation, and cancer: respirable particulate matter, fibrous dusts, and ozone as major causes of lung carcinogenesis through reactive oxygen species mechanisms.	Int J Environ Res Public Health;10(9):3886–3907	Telomere shortening/oxidative/epigenetic effects	Unidentified	Review
Oit-Wiscombe. 2013 (134)	Increased DNA damage in the progression of COPD: a response by poly(ADP-ribose) polymerase-1.	PLoS One; 8(7):e70333	Oxidative	BER	Human
Silva ALG. 2013 (86)	Evaluation of DNA damage in COPD patients and its correlation with polymorphisms in repair genes.	BMC Medical Genetics;14:93	Polymorphisms/oxidative/Double strand break	BER, HR, NHEJ	Human
Kim WJ. 2014 (125)	Association of lung function genes with chronic obstructive pulmonary disease.	Lung;192(4):473–480	COPD susceptibility	Unidentified	Human
Yang S. 2014 (135)	Feasibility of 8-OHdG formation and hOGG1 induction in PBMCs for assessing oxidative DNA damage in the lung of COPD patients.	Respirology; 19:1183–1190	Oxidative	BER	Human
Korytina GF. 2014 (136)	Association of Genes Involved in Nicotine and Tobacco Smoke Toxicant Metabolism (CHRNA3/5, CYP2A6, and NQO1) and DNA Repair (XRCC1, XRCC3, XPC, and XPA) with Chronic Obstructive Pulmonary Disease.	Mol Biol;48(6):823–883.	Polymorphisms/oxidative	NER	Human

There are different DNA repair pathways, but one of them is mainly related to oxidative damage. The excision repair, also known as “repair in the dark,” was described in 60's by Setlow and Carrier, when they observed DNA repair could carry out by another mechanism, which had not been described yet. This DNA repair occurs when the damaged nitrogenous base is removed and replaced by a normal base in the DNA molecule. Excision repair can be divided into two mechanisms: base excision repair (BER) and nucleotide excision repair (NER) (61).

DNA repair for specific oxidative damages by base excision repair mechanism

Reactive oxygen species (ROS) react with DNA molecule causing simple strand breaks (SSB), especially in sugar bonds (62). 8-oxo-guanine (8-oxoG) and 5-hydroxycytosine, which mispair with adenine and thymine, respectively, are the most commonly oxidized bases (63). As previously described, COPD contributes to increase in free radicals, thus causing oxidative stress, which in turn could damage the DNA molecule and consequently activate base excision repair mechanism (64). Oxidative damage is increased in tumors, thus increasing metabolism, oncogenic signaling, and mitochondrial dysfunction (65). This factor results in 100 times more 8-oxoG in cancer tissues than in normal tissues (65).

Base excision repair acts on single strand breaks, oxidized bases, and little bulky adducts (66), induced by free radicals that produced by endogenous mechanisms and those induced by exogenous agents (64). This mechanism is evolutionarily conserved, with high functional sequence homology from bacteria to mammals presenting two pathways: short-patch BER pathway repairs a single base, and the long BER pathway is able to remove from two–eight bases (67) (Figure 2A). BER is characterized by excision of the damaged base by a series of enzymes called DNA glycosylases (68). 8-oxoguanine DNA glycosylase (OGG1) is a glycosylase, which recognizes oxidized bases, promotes hydrolysis of N-glycosyl bond, and also contains lyase activity (68). Consequently, there is a formation of an abasic site, which is recognized by an apurinic/apyrimidinic endonuclease (APE, in mammalian cells are APE1 and APE2), which in turn produces breaks in the phosphodiester bond 5′ or 3′ in the abasic site (69). After the action of the glycosylase, with the formation of the AP site and the disruption of the DNA strands, the Poly (ADP-ribose) polymerase 1 (PARP1) performs the process known as “PARylation,” in order to modify the surroundings of chromatin proteins, the polarity of the DNA strand, which contributes to the recruitment of other proteins to the BER (69).

Figure 2. Schematic representation for DNA repair by base excision mechanism. (A) Schematic representation for base excision repair mechanism (REB) by short patch (for one damaged base) and long patch (for up to eight damaged bases). (B) Schematic representation for global genome and transcription-coupled nucleotide excision repair mechanism (REN). Endo III (endonuclease III), OGG1 (8-oxoguanine DNA glycosylase) MPG (3-methyladenine-DNA glycosylase), Xth (xyloglucan endo-transglycosylase/hydrolase), XRCC1 (X-ray repair cross-complementing protein 1), PCNA (proliferating cell nuclear antigen), Lig (ligase), XP (xeroderma pigmentosum), ERCC1 (excision repair cross-complementing 1), RPA (replication protein A) and Lig3 (ligase III).

The interaction between Rapamycin and Torin 1 (RAP) protein and PARP1 with X-ray repair cross-complementing (XRCC1) protein stimulates APE1 required for the cleavage of the phosphate backbone, thus generating an AP site, and creating a single-strand break. After cleavage, the polymerase (POL) inserts the new nucleotide (68).

The gap formed is therefore filled with a new nucleotide by action of DNA polymerase β (POL β). Additionally, this enzyme presents phosphodiesterase activity, which hydrolyzes unmodified 5 ends, thereby subsequent connection of the new nucleotide to DNA sequence involved by DNA ligase III that steals the DNA ends (69). BER is not a flexible mechanism for the recognition of DNA damages, since it is not feasible for cells to maintain different enzymes for different types of damages encountered in the DNA (70). The switch of short or long pathway could be linked to the availability of ATP in the vicinity of the AP site (71). In this context, NER is more general and flexible, being able to recognize and repair a large number of damages, not necessarily those related to the DNA structure.

DNA repair for non-specific oxidative damages by base excision repair mechanism

As discussed for BER, COPD contributes to increase in free radicals, which in turn could instigate oxidative stress and consequently damage the DNA molecule; thus nucleotide excision repair mechanisms may be activated and effectively contribute to DNA molecule repair (72). Recognition of DNA damages by NER does not occur by their chemical nature, but by the degree of distortion promoted in the DNA helix. DNA damages, as cyclobutane pyrimidine dimers, 6–4 pyrimidine-pyrimidone chemicals adducts, and certain types of cross-links between the two strands of DNA, caused by substances found in tobacco smoke and food, and intrastrand crosslinks formed by chemotherapeutics like cisplatin are the main cause of DNA deformation (73,74).

Nucleotide excision repair removes 22–30 nucleotides surrounding a helix distorting DNA damages, and depending on the distortion in the double strand, damages are repaired more efficiently or not, but usually occur evenly in the whole genome (75,76). This mechanism could be involved in the repair of some damage types, such as oxidized bases, but its role in the repair of oxidative damages is not completely elucidated (72).

More than 30 different proteins are involved in the NER mechanism, presenting two pathways: global genomic repair, which removes the damages in non-transcribed regions, and transcription coupled repair, which removes damages at transcriped genes (61) (Figure 2B). In the global genomic repair pathway, after the primary recognition of a wide variety of damages in the DNA molecule by the complex XPC/HR23B, factors are recruited, whereas in the transcription coupled repair, the above-mentioned complex is essential for the recognition of the damage (74,77).

However, blocking RNA polymerase II (RNAPII) by damaged DNA, such as those induced by free radicals, is considered as an induction signal for DNA repair mechanisms. After damage recognition, RNAPII enzyme is removed by CSA and CSB proteins (“Cockayne Syndrome” A and B), and DNA repair is started (78). Xeroderma pigmentosum complementation group E (XPE) protein complex, whose subunits are P127/DDB1 and p48/DDB2, recognizes damages not identified by the XPC complex (79). After detection, both NER mechanisms converge to a common pathway for the cleavage of the DNA double strand. RNA polymerase II transcription factor (TFIIH - “Transcription factor II Human”) is recruited to the damage site, the TFIIH may be able to carry out a 5′ to 3′ search for a short distance before anchoring at DNA damage in cooperation with the other protein components of the preincision complex, and provides a catalytic activity to open the DNA (80). TFIIH 9 is composed of subunits of ATP (p62, p52, p44, p34, cdk7, cyclin H and MAT 1). In addition to these subunits, 2 helicases, called XPB and XPD, participate in NER. The role of complex proteins, and the XPD and XPB proteins are present at the opening and unfolding of the DNA double helix at the site of damage, thereby allowing the entry of the NER factors that will succeed (81).

The ATP-dependent helicase 5′ to 3′ (XPD/ERCC2) is required for NER, differently from XPB, whose action is not essential. The role of helicase XPD/ERCC2 is also important to maintain the stability of the complex as a whole, through the interaction of the C-terminal portion with another p44 subunit complex. Some studies suggest that XPD is involved in DNA repair after the recognition of the double helix distortion by the XPC/HR23B complex, checking if there is indeed the presence of damage (76,82).

The complex constituted by Repliacation Protein A (RPA), XPA and XPG proteins conducts the pre-incision, and performs the DNA repair synthesis. The incision in DNA molecule is carried out by a heterodimer constituted by the XPF and Excision Repair Cross-Complementation group 1 (ERCC1) protein, in addition to XPG enzyme. XPF-ERCC1 heterodimer is recruited to the damage site over XPA protein to generate the incision, which occurs in the nucleotide sequence about 15–24 nucleotides at the 5′ side and about 2–8 nucleotides at the 3′ side of the damage (83).

During the 3′ cleavage of the DNA strand by ERCC1-XPF complex, the synthesis of the new DNA fragment is started before a 5′ cut of the DNA strand by the XPG protein (78). The XPF-ERCC1 complex, cited above, is part not only of the NER mechanism, but also of other DNA repair pathways, such as the repair of double breaks of DNA, cross-links, and it is essential for the development in humans as well (76,84).

Nuclear Sirtuin 1 (SIRT1) protein plays a dual role in promoting NER. Firstly, this protein stimulates the recognition of damage by promoting the expression of XPC protein (76) and then, stimulates the excision of damage promoting the NER endonuclease assembly at damage sites by deacetylation of XPA protein (85). DNA synthesis repair is carried out by RPA, RFC, PCNA, and polymerases σ/ϵ, while reclosing of the strand is carried out by the DNA ligase, either LIG3 or LIG1 (61).

Other types of DNA repair in COPD

Some studies suggest that patients with COPD present double-stranded breaks in the DNA molecule (86), and also that tobacco consumption leads to DNA strand double breaks in leukocytes (87). This damage induces phosphorylation of histone γ-H2AX (H2AX), which in turn recruits other DNA repair proteins, such as Breast Cancer-Associated 1 (BRCA1) protein, resulting in the activation of downstream distinctive repair pathways (88).

Starting from the double breaks, DNA will be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ) repair mechanisms. HR acts preferentially during the S phase, while NHEJ, the main repair mechanism to repair double-strand breaks in higher organisms acts throughout the cell cycle phases (89).

Homologous recombination presents three stages or phases: presynapsis, synapsis, and postsynapsis. Homologous DNA without damages is required to repair the DNA molecule. (90) (Figure 3A). In the presynapsis stage there is a 5′ to 3′ resection process of broken DNA ends by complex to generate to generate a 3′ single ended strand DNA by removing about 100 nucleotides with interaction with the C-teminal protein (CTIP) (91). This MRN heterotrimeric complex is formed by Mre11, NBS 1, and Rad 50 proteins and the MRX heterotrimeric complex is formed by Mre11, Rad 50, and XRS2, which performs the unwinding of the DNA end, while recruiting the BRCA1 protein, whose function is to make an incision and promote activation of CTIP (92).

Figure 3. Schematic representation for DNA repair by recombination mechanism. (A) Schematic representation for homologous recombination. (B) Schematic representation for non-homologous end-joining after DNA double-strand break. DNA PKcs, DNA-dependent protein kinase, catalytic subunit; XRCC4, X-ray repair cross complementing 4; XLF, XRCC4-like factor; ATM, ataxia-telangiectasia mutated; RPA, replication protein A.

In the synapsis stage, heterotrimeric RPA binds at the single-stranded DNA to prevent internal base pairing (92). Then, RPA and Rad 51 proteins are replaced by recombinase complexing with BRCA2 and paralogs of Rad 51 (RAD51B, C and D, XRCC2 and XRCC3) and the DNA filament will be target of nucleoprotein Rad51 for nucleoprotein Rad51 (93), which identifies homologous sequences in DNA molecule. Thus, Rad51 leaves the 3′ end to reveal a 3′-OH group, permiting that DNA synthesis enzymes access the DNA end (94). From this stage, DNA repair process could take place by the synthesis-dependent strand-annealing (SDSA) or the double-strand break repair (DSBR) pathway.

In the SDSA pathway, after DNA synthesis is performed by polymerase δ, the new DNA fragment is moved and reconnected with the original strand. In the DSBR pathway, two independent strand invasions from both DSB ends are followed by simultaneous DNA synthesis performed by polymerase η (95).

On the other hand, the NHEJ mechanism acts on double-strand breaks reconnecting the two DSB ends. Different from HR, this mechanism can connect any type of DNA end without homology between the sequences. However, NHEJ is more susceptible to errors, once this mechanism can cause small insertions or deletions of nucleotides in the DNA sequence (96).

Double-strand break DNA repair by NHEJ starts with the regulatory subunits binding to DNA, Ku70 (also known as XRCC6), and Ku80 (also known as XRCC5), along with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (Figure 3B). DNA-PKcs is a member of the PI3K-related protein kinase (PIKK) family, which also includes ataxia-telangiectasia (ATM), Rad3-related and Mechanistic Target of Rapamycin (mTOR) proteins. Assembly of these proteins form the DNA-PK holoenzyme, and are involved in the recognition of the damage. Pol μ and λ participate in the pathway by binding to Ku proteins by N-terminal domain breast carboxy-terminal cancer (CRCT), providing their gap-filling activity before the end ligation step, which is performed by the XRCC4-ligase IV complex (97). While Pol λ synthesizes new DNA with partially complementary ends, Pol μ synthesizes DNA without the presence of complementary strand (98).

DNA-PK enabled recruit other proteins, such as Artemis (which solves the DNA ends), XRCC4, XLF, DNA ligase IV, and DNA polymerase, which complete the repair (99). Double-strand breaks could trigger permanent cell cycle arrest or apoptosis, leading to important clinical changes, including carcinogenesis.

DNA damages, mainly those caused by oxidative processes, are related to some chronic diseases, such as diabetes mellitus, atherosclerosis, neurodegenerative diseases, and cancer (100). Papi et al. (101) in a case report suggested that COPD, with the exception of chronic bronchitis, increases up to 3 times the risk of patients developing squamous cell carcinoma by the activation of signaling pathways (101). These signaling pathways could be activated by the peripheral distribution of tobacco due to the limitation of the air flow, but other studies reinforce that the activation could also occur by second messengers, as ROS (102). Caramori et al. (103) suggested that inefficiency or imbalance of DNA repair, such as the reduction of expression of one of the Ku86 DNA repair proteins in bronchiolar epithelial cells of patients with COPD, could contribute to increased risk of lung carcinoma (104). Also, a strong correlation between increased oxidative stress and DNA damage and cancer development had been observed, suggesting that molecular studies aiming at new targets for carcinoma prevention are necessary (105). Moreover, molecular targets should take into account not only individuals with COPD, but also at-risk groups, such as smokers, reinforcing that it is necessary to understand the molecular interactions in the development of COPD to achieve molecular targets, which are not present in clinical trials yet (106).

Telomeric and genomic stabilization followed oxidative DNA damage

A number of studies show that DNA damages cause genomic instability, shortening the telomeres, for example. Mammalian telomeres are composed of repeatitions of TTAGGG sequences, as a t-loop, followed by a 3′ end, and they are related to their self-protection and cell longevity (107). During normal cell divisions, enzymes (telomerases) act shortening the telomeres (108). In oxidative stress conditions, such as in those with COPD (109), a drastic telomere shortening has been reported (110). Interestingly, this telomere shortening was related to chronic inflammation in COPD patients (111). Telomere shortening in COPD is related to reduced telomerase activity (111). A study showed that the predisposition to lung emphysema could cause telomere shortening induced by lung injury due to cigarette smoke (112). Another study demonstrated that telomere shortening is associated to increased risk of COPD, as well as to reduction of lung function (113).

In addition to telomerase, telomere integrity relies on shelterin, a DNA-bound protein complex composed by six polypeptides (TRF1, TRF2, RAP1, TIN2, TPP1, and POT1) (114) (Figure 4). Repeat-binding telomeric factors 1 and 2 (TRF1 and TRF2, respectively) directly bind at the double-stranded telomere repeats, POT1 binds directly at the single-stranded 3′-overhang, and the other three components interconnect these telomere-binding components. TRF1 and TRF2 interact with other proteins (115) in order to protect the telomeres by directly binding to the telomeric duplex (116). The interaction between TRF1 and TRF2, mediated by TRF1-interacting factor-2 (TIN2), contributes to the stabilization of TRF2 on telomere, protecitng DNA ends from degradation (117).

Figure 4. Schematic representation for protein performance in telomeric regulation and genomic stabilization mechanisms. TRF, telomeric repeat-binding factor; TIN2, TRF1-interacting factor-2; POT1, protection of telomeres 1, TPP1, tripeptidyl-peptidase 1; ATM, ataxia telangiectasia mutated; p53, tumor protein 53; MDM2, mouse double minute 2 homolog.

Telomere deprotection, as a consequence of shelterin loss or telomere erosion, induces activation of Ataxia Telangiectasia (ATM) protein and inactivation of Tumor protein p53 (Tp53), which result in chromosome fusions by NHEJ and/or in apoptosis (118). These factors regulate the telomere protection, telomere length, and interact with DNA repair proteins involved in chromosome instability syndromes, which cause premature aging, as well as increased cancer risk (119).

A study suggests that TRF2 levels and telomere protection are subject to modulations by MAP Kinase signaling, such that cell growth could require to be coupled to TRF2-dependent telomere functions in order to ensure proper cell division (120). Other studies have suggested that the loss of TRF2, for example, causes activation of ATM-dependent DNA damage response and DNA repair by NHEJ, resulting in p21-mediated G1/S arrest and downregulation upon p53 activation (119) (Figure 4). The p53 protein acts as a tumor suppressor, participating in some cellular processes, such as cell cycle regulation, apoptosis, and DNA repair (121). p53 inactivation aborts the TRF2-ATM-p53 feedback regulation (122), inducing cancer-promoting conditions, suach as those in lung cancer. In these conditions, dysfunctional telomeres with supra-physiological amounts of TRF2 are susceptible to chromosomal instability, and senescence checkpoint is impaired (123).

Chronic obstructive pulmonary diseases have a prevalence in the elderly people, and have been related with telomere shortening (124). Additionally, in oxidative stress there is a significant acceleration of telomere shortening (110), and shortness of telomere length was indicated to be associated with a 28-fold increased risk of COPD (113). Although age contributes to the onset of COPD, shortening of telomeres is associated with increased risk of the prevalence of these diseases (94).

There are not studies correlating the development of COPD directly with telomere instability. However, after the initiation of COPD, DNA damage is commonly associated with oxidative stress, and could be used as a diagnostic marker (59). COPD patients have a high incidence of lung cancer as previously mentioned, and DNA modifications by reactive species could be the relationship between these two conditions (59). Also, it is not reported if COPD is induced by total or partial absence of enzymes involved in the repair of DNA molecule. However, due to a series of studies that show mainly oxidative damage to DNA, it is possible to suppose that repair mechanisms have a key role in the correction of damage, thus contributing to a better prognosis in COPD. In fact, a study suggests that deficiency in DNA repair in response to oxidative stress could lead to COPD, lung cancer, or pulmonary fibrosis (5).

Conclusion

In COPD, inflammatory process is intense, increasing the oxidative stress mediated by free radicals and DNA damages. Although there are progresses in treatment for COPD, repair of DNA damages could be important against COPD development, and therapies based on the modulation of DNA integrity and genomic stability could be effective to reduce the mortality and morbidity of patients with COPD.

Declaration of interest

The authors report no conflicts of interest.

Funding

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).