Interstrand Crosslink Repair: New Horizons of DNA Damage Repair

*Amna Aqeel, Javaria Zafar, Naureen Ehsan, Qurat-Ul-Ain, Mahnoor Tariq and Abdul Hannan*

## **Abstract**

Since the dawn of civilization, living organisms are unceasingly exposed to myriads of DNA damaging agents that can temper the ailments and negatively influence the well-being. DNA interstrand crosslinks (ICLs) are spawned by various endogenous and chemotherapeutic agents, thus posing a somber menace to genome solidity and cell endurance. However, the robust techniques of damage repair including Fanconi anemia pathway, translesion synthesis, nucleotide excision and homologous recombination repair faithfully protect the DNA by removing or tolerating damage to ensure the overall survival. Aberrations in such repair mechanisms adverse the pathophysiological states of several hereditary disorders i.e. Fanconi Anemia, xeroderma pigmentosum, cerebro-oculo-facio-skeletal syndrome and cockayne syndrome etc. Although, the recognition of ICL lesions during interphase have opened the new horizons of research in the field of genetics but still the detailed analysis of conditions in which repair should occur is largely elusive.

**Keywords:** DNA damage repair, Interstrand cross links (ICLs), Homologous Recombination Repair, Translesion synthesis, Non-homologous end-joining repair, FA pathway

### **1. Introduction**

There is an amalgam of various environmental, endogenous as well as chemotherapeutic agents that are continuously having a contact with the genetic material in living beings and making it a point of real concern throughout the globe. The attack of reactive oxygen as well as nitrogen species on DNA have contributed towards a large amount of defects and complex chemical structures that take place in DNA [1]. These damages give rise to a series of simple and bulky base modifications that distort the helical structure, abasic sites, the breaks in phosphodiester linkages along with the interstrand crosslinks (ICLs). These lead to various mutagenic changes in the genetic blueprint and become a reason of inhibition of the transcriptional or replicative machinery that induce activate apoptotic divisions or necrosis [2].

Interstrand cross-links (ICLs) are the anomaly that link the complementary strands of DNA by the covalent linkage between the bases. These are formed by the chemicals along with the two reactive electrophilic groups. It is a highly sequence-dependent reaction in which the two nucleophilic groups on the opposite strands are aligned geometrically and enable the dual reaction of the bifunctional cross-linking agent with it. This complex chemical reaction give rise to ICLs,

mono-adducts, intrastrand cross links as well as DNA-protein cross-links [3]. The ICLs are made with the help of reactive endogenous chemicals such as lipid peroxidation product known as malondialdehyde or aided with the reactive aldehyde group of an unpromptedly formed or the enzyme-derived abasic site in the DNA molecule with a normal base on the complementary strand [4].

A large amount of anticancer and chemotherapeutic agents such as mitomycin C (MMC), cisplatin, nitrosoureas and nitrogen mustards are notorious for introducing formidable blocks in the normal metabolic processes of DNA with ICLs and need repair for cell sustenance. ICLs are also caused by various antitumor agents that defects DNA through radical processes like C-1027, neocarzinostatin [5]. With the passage of time, the organisms have developed various complex mechanisms to alleviate these deleterious defects from the genome. The failure to remediate the defect can contribute towards cell death that can occur either through a mitotic catastrophe or the p53-dependent apoptotic pathway. In the mammalian cells, the repair mechanisms for ICLs repair are still ambiguous [6]. According to an estimation, about 40ICls that form in a mammalian genome can destroy a defective cell that lacks ability to be repaired.

The in vivo study gives an overview of the elimination of the ICLs in cellular DNA of both prokaryotes and eukaryotes. The model organisms are used for the clear understanding of the repair mechanisms. These include *E. coli* and yeast. The ICLs repair mechanisms in bacteria and yeast are replication dependent and independent while in vertebrates, they follow repairment pathway during replication of DNA [7]. Moreover, the recent study suggests the operation of replication independent ICL repair pathway in vertebrates.

The ICL repair pathway have been deduced from the relative sensitivity of the DNA repair defective cell lines to the cross linking agents. Pathways of ICL repair have mostly been inferred from the sensitivities of DNA repair defective cell lines to crosslinking agents. During the S phase of the cell division in vertebrates, the ICL repair is induced by the help of impeded replication forks. The process of ICL repair needs a nexus of multiple factors along with the structure specific endonucleases, for example TLS and HR. If a disturbance occurs during the repair, the genomic instability results that bring forth the birth of Fanconi anemia, a cancer prone ailment [8]. There is another ICL repair pathway that takes place in the G0/1 phase during the cell cycle which is a replication and recombination independent pathway [9]. In addition, the tolerance of ICLs in G1 as compared to S phase makes it an underappreciated pathway because there, the stalled replication fork possesses high toxicity. Contrarily, the toxicity of ICL in G1 can be depicted when it terminates the transcription of a gene playing a vital role.

The latest studies have proposed the role of NER proteins (as they cut one side of ICL) [7], Homologous recombination along translesion synthesis polymerases (Polζ, Rev1) that are involved in filling the gap for both type of cells undergoing replication as well as non-replicating ones [10]. The proteins involve in the ICL repair have a vital role in the pathophysiology of several hereditary diseases Proteins implicated in the repair of ICLs have a critical role in the pathophysiology of several hereditary disorders. In addition, cells deficient in the Fanconi Anemia (FA) pathway are highly sensitive to ICLs [11] and this pathway has been suggested to play an important role in mammalian ICL repair at replication forks promoting homologous recombination. There has been a series of continuous research on ICL lesions in the past decade and it covered the various aspects of ICLs be it as their identification, detection methods or their development along with the repair mechanisms and the exploitation of cross linkers in the laboratory. These have paved the way towards the better and more reliable understanding of ICLs in the complex biological samples. This chapter foregrounds the multiple aspects of the interstrand cross-link repairs with a reference to their pathophysiology and lesion repair mechanisms.

**123**

*N*2

*Interstrand Crosslink Repair: New Horizons of DNA Damage Repair*

as well as exogenous sources of ICLs are summarized as follows:

A large variety of natural and synthetic chemicals are notorious for bringing ICLs on the front and are regarded as the ICL inducers or inducing agents. In the same way, the metabolic byproducts formed in the cell also contribute towards ICLs formation. Their structure and function vary greatly but ICLs inducers are known for their bifunctional reactivity with both of the strands of DNA. The endogenous

The endogenous sources of ICLs comprises of the reactive aldehydes that are generated as a result of lipid peroxidation along with base excision repair (BER) [12]. There are other endogenous by products of lipid peroxidation, the α, β-unsaturated aldehydes or enals namely crotonaldehyde, acrolein, along with the 4-hydroxynonenal (4-HNE). These are formed as a result of oxidative stress [13]. Moreover, there are exogenous contributors as well namely cigarette smoke and automobile exhaust to expose with acrolein and croton-aldehyde. The DNA nucleobases interact with enals to give rise to exocyclic adducts. These adducts then interact with proteins. The incorporation of enals to dG is done with the

(3-oxopropyl)-dG adducts. The next stage is cyclization of N1 with the aldehyde,

genotoxic to human beings. Shapiro and Leonard are famous for their earlier study of nucleosides reactions with glyoxal, chloroacetaldehyde, malondialdehyd along with related bis-electrophiles [14, 15]. The in vitro formation of ICL is attributed to

Moreover, there are DNA lesions that are formed as a result of accumulated acetaldehyde in the cells. The acetaldehyde is produced as a result of alcohol metabolism with aldehyde dehydrogenase 2 (ALDH2) as a biocatalyst. The drug disulfiram if used, blocks the enzyme ALDH2 and accumulates the acetaldehyde in the cells. The lesions produced are DNA adducts, breaks in single or double-strands of DNA (DSBs), sister chromatid exchanges (SCEs), point mutations, along with

etheno-2′-deoxyguanosine are vital DNA damage agents that follow the accumulation of acetaldehyde in the cells. The acetaldehyde reacts with guanine and forms

with N2 amine of guanine in 5′-CpG sequence consequently forming acetaldehyde interstrand crosslinks (AA-ICL). In Asian continent, the irreparable detoxification of acetaldehyde is found more often and is linked with alcohol mediated cancers [18]. Moreover, cells in *Saccharomyces cerevisiae* don't have ability to repair ICLs and are acetaldehyde sensitive thus gives validation of acetaldehyde mediated ICLs [19]. The intestinal pathogens in human beings known as *Enterobacteriaceae* and other bacteria play a vital role in the progression of colorectal cancer. They produce colibactins that are genotoxic in nature and bring harm to human beings. With their structural chemistry still unknown, colibactins produce ICL dependent DNA double-strand breaks (DSBs) and activates the ICL repair pathways [20]. *Cellulo* also depicts another picture of the DNA damaging mechanism in which colibactin producing bacterial exposure towards the genomic DNA of cultured human cells made it susceptible to interstrand cross links. There are different changes observed in the intoxicated cells including the replication stress, the activation of









**2. Basic biochemistry of ICL-generating agents**

**2.1 Endogenous sources of interstrand cross links**

help of Michael addition in which addition of N2

and forms ICL on exposure towards an aldehyde [16].

crosslinks in DNA [17]. The DNA adducts like *N*<sup>2</sup>


a crosslink precursor known as N2

the opening of the exocyclic 1, N2

giving rise to N2

*DOI: http://dx.doi.org/10.5772/intechopen.97551*
