MECHANISMS OF DNA DAMAGE AND REPAIR

Living organisms are continuously exposed to a myriad of DNA-damaging agents that can impact health and modulate disease states. However, robust DNA repair and damage-bypass mechanisms faithfully protect the DNA by either removing or tolerating the damage to ensure overall survival. Deviations in this fine-tuning are known to destabilize cellular metabolic homeostasis, as exemplified in diverse cancers where disruption or deregulation of DNA repair pathways results in genome instability. Because routinely used biological, physical and chemical agents impact human health, testing their genotoxicity and regulating their use have become important. In this introductory review, we will delineate mechanisms of DNA damage and the counteracting repair/tolerance pathways to provide insights into the molecular basis of genotoxicity in cells that lay the foundation for subsequent articles in this issue.

Introduction

Preserving genomic sequence information in living organisms is important for the perpetuation of life. At the same time, mutagenesis plays an indispensable part in its maintenance and evolution, while also contributing to cancer, certain human diseases, and aging. It is known that DNA, the basic unit of inheritance, is an intrinsically reactive molecule and is highly susceptible to chemical modifications by endogenous and exogenous agents. Furthermore, the DNA polymerases engaged in DNA replication and repair make mistakes, thereby burdening cells with potentially disadvantageous mutations. However, cells are equipped with intricate and sophisticated systems—DNA repair, damage tolerance, cell cycle checkpoints and cell death pathways—that collectively function to reduce the deleterious consequences of DNA damage.

Cells respond to DNA damage by instigating robust DNA damage response (DDR) pathways, which allow sufficient time for specified DNA repair pathways to physically remove the damage in a substrate-dependent manner. At least five major DNA repair pathways—base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR) and non-homologous end joining (NHEJ)—are active throughout different stages of the cell cycle, allowing the cells to repair the DNA damage. A few specific lesions can also be removed by direct chemical reversal and interstrand crosslink (ICL) repair. These repair processes are key to maintaining genetic stability in cells. In addition, certain types of DNA damage are substrates for the DNA damage tolerance pathways. In higher eukaryotes, for example, a well-orchestrated group of five main translesion synthesis (TLS) polymerases—REV1, POL ζ, POL η, POL κ and POL ι—bypass the damage to enable the continuation of replication, but with the possibility of a concurrent introduction of an incorrect base that can be fixed into a mutation in the subsequent round of replication. Under the circumstances, when the damaged DNA persists, programmed cell death or apoptosis, a regulatory response to DNA damage, is activated to get rid of cells with extensive genome instability.

Not surprisingly, in many cancers, DNA repair, DNA damage tolerance, and DDR pathways are disrupted or deregulated, which increases mutagenesis and genomic instability, thereby promoting cancer progression. Likewise, aging is attributed to the attrition of chromosomal ends and the failing capacities of a combination of these pathways. Other diseases, such as neurodegenerative disorders, result from a combinatorial failure of more than one of these processes. The 2015 Nobel Prize in Chemistry to Drs. Lindahl, Modrich, and Sancar highlight the importance of mechanisms of DNA damage and repair and their implications for human health. In this review, we will discuss the details of various types and mechanisms of DNA damage and the compensatory repair and tolerance pathways.

Methods

DNA damage can be categorized into two main classes based on its origin: endogenous and exogenous. The majority of endogenous DNA damage arises from the chemically active DNA engaging in hydrolytic and oxidative reactions with water and reactive oxygen species (ROS), respectively, that are naturally present within cells. Such inherently predisposed reactions of DNA with molecules from its immediate surroundings fuel the development of hereditary diseases and sporadic cancers. Exogenous DNA damage, on the other hand, occurs when environmental, physical, and chemical agents damage the DNA. Examples include UV and ionizing radiation, alkylating agents, and crosslinking agents. We offer here a brief summary of the main endogenous and environmental agents that produce the different classes of DNA damage that then become substrates for the specific DNA repair pathways discussed in the subsequent section.

Discussion

In the last decades, understanding the genetic bases of many human disorders has made unprecedented steps forward. This has fostered the development of novel therapeutics aimed at curing patients affected by genetic defects by means of gene therapy. This typically includes the development of viral vectors transferring a correct copy of the mutated gene. Once present in the host cell, the expression of the correct gene complements the missing gene function in the patient cells, resulting in a functional cure. With the dawn of targeted genome editing, the chase for new approaches aimed at precisely correcting the disease-causing mutation was launched. In this case, the first step relies on the introduction of a DSB in close proximity to the genomic site where the change is desired. This is made possible by using programmable designer nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the recently introduced CRISPR-Cas (clustered regularly interspaced palindromic repeat-CRISPR-associated) systems. For a comprehensive description of the different types of designer nucleases, we refer the reader to detailed reviews published elsewhere. As described in the previous sections, the cells have developed sophisticated mechanisms to sense and resolve DNA lesions, such as DSBs, in order to maintain genome integrity. The precise insertion of a DSB by such programmable nucleases triggers either of the two major repair mechanisms, NHEJ or HDR, which can be harnessed to achieve precise and permanent changes in the human genome.

Conclusion

Understanding how genome stability is maintained and investigating the mechanisms adopted by the cells to withstand DNA lesions has been paramount to exploring the therapeutic potential of genome editing. We have illustrated how failure in DNA repair mechanisms may contribute to the onset of detrimental conditions, such as cancer. Moreover, we have described how this knowledge is exploited to develop new therapeutics based on “synthetic lethality” or genome editing using designer nucleases. While the activity of designer nucleases at sites that share certain sequence identities with the target site, the so-called off-targets, poses concerns, the increasing number of genome editing trials approved thus far and the first in human application of CRISPR-Cas to treat a blindness disorder recently described suggest that new therapeutics are on the horizon. Once current approaches are substantiated in conditions that better resemble those of transplanted patients and genome-wide analysis to dissect the genotoxic potential of these approaches are in place, it will be reasonable to believe that new treatment opportunities will be available for more and more human disorders using a new generation of therapeutics.