Cell Cycle 101: DNA Proofreading and Repair Mechanisms
Foreword
Have you ever questioned how a wound heals? How does an embryo become a baby? How does a cancerous tumor grow? At the heart of these questions lies cell replication and division. The remarkable ability of a cell to grow and divide into two identical daughter cells underpins the success of life. This is, however, only conceivable because eukaryotic cells (that is, with nuclei) go through the cell cycle, a set of well-defined and tightly synchronized events. This process is divided in two main stages: the interphase, whereby a cell grows; and the mitotic phase, by which cells replicate (mitosis) and divide (cytokinesis). During this process deoxyribonucleic acid (DNA), also recognized as the "molecule of life," is accurately replicated and faithfully distributed to newly generated daughter cells. DNA is the molecule that carries and conveys hereditary material or genetic information from parents to offspring, thus holding all the information needed to construct and sustain an organism. The cell cycle thus functions as the master regulator of tissue growth, development and proliferation. It also ensures that old cells are removed in a timely manner as we age and replaced by new cells that proliferate in a tightly controlled fashion. This keeps the total number of viable cells constant, which is essential for life as we know it.
The Cell Cycle 101 series attempts to elucidate the most important aspects of the cell cycle: from its history and discovery to the underlying mechanisms that control cell division. This series aims to explore the intricacies of cell cycle control in development and the consequences of its failure in disease.
Therefore, this series is divided into the following chapters:
6. Cell Cycle 101: DNA Proofreading and Repair Mechanisms
Cell Cycle 101: DNA Proofreading and Repair Mechanisms
Considering DNA is the reservoir of genetic information within every functioning cell, maintaining its integrity and stability is critical to life. While preserving genomic sequence integrity is required for the maintenance of life, the engine of evolution is mutagenesis, the process by which an organism's genetic information is altered through mutations that lead to diversity. The main drawback of mutation, however, is that it can contribute to cancer, certain human diseases, and aging (Chatterjee & Walker, 2017). DNA is not inert, but rather a reactive molecule, susceptible to chemical modifications by endogenous and exogenous agents that can cause damage (Clancy, 2008). DNA damage is caused by the integration of faulty nucleotides (the structural units of DNA) during replication, as well as chemical changes caused by spontaneous mutations or exposure to environmental factors such as radiation. If the ensuing damage is not repaired, it can lead to mutation, i.e., irreversible changes in the DNA sequence, and in certain cases to disease (Clancy, 2008; Hakem, 2008). Skin cancer, triggered by UV radiation from sunlight, is perhaps the most widely recognized example of the close relationship between environmental DNA damage and disease (Yu & Lee, 2017). Another example is the damage produced by smoking tobacco, which can lead to lung cell abnormalities and eventually lung cancer. Aside from environmental factors, DNA oxidative damage is also caused by by-products of cellular metabolism. In fact, it has been estimated that a single cell can undergo up to a million genetic changes per day (Lodish et al., 2004). In most cases, however, this damage does not lead to cancer or mutations. This is because DNA proofreading and repair mechanisms locate and fix errors in the DNA code. If this damage is irreparable, the cell undergoes programmed cell death (apoptosis) in order to avoid passing on the faulty genetic information to the next generation (Bębenek & Ziuzia-Graczyk, 2018; Clancy, 2008). Cancer, on the other hand, develops when these repair systems fail, allowing multiple alterations in division-related genes to accumulate in a single cell. Cells that have acquired such mutations proliferate rapidly, disregard normal stop signals, and form a tumor.
DNA Proofreading and Repair
DNA damage has both immediate and long-term repercussions. The process of DNA replication itself carries a high probability of yielding errors that can eventually develop into mutations. These arise from errors made by the enzymes engaged in DNA synthesis (DNA polymerases), thereby burdening cells with potentially detrimental mutations. However, DNA polymerases themselves check their work as each nucleotide is being added, acting as repair systems (Hoeijmakers, 2001). This kind of proofreading consists of a spell-checking-like mechanism that allows DNA polymerases to recognize and remove newly formed nucleotides that have been incorporated incorrectly. This is only possible because the polymerase, when adding a nucleotide to the new strand, checks whether its base is complementary to the nucleotide of the original strand. There are four nucleotide base options: adenine (A), thymine (T), cytosine (C) and guanine (G); and each base can only bind to each other, A to T and C to G, a rule known as complementary base pairing (Bates, 2023). Before DNA replication can proceed, the faulty nucleotide is immediately removed and replaced with the correct one. Proofreading with DNA polymerase increases replication fidelity by 100-fold, which is required by many species to prevent excessively high, life-threatening mutation rates. Some mutations, however, escape this mending process (Bębenek & Ziuzia-Graczyk, 2018; Reha-Krantz, 2010).
To counteract DNA damage, cells are equipped with intricate and sophisticated systems known as DNA damage response (DDR) pathways that collectively function to reduce its deleterious consequences. A well-coordinated network of genome maintenance mechanisms shields DNA from damage or helps mitigate its negative effects. Throughout evolution, a complex arsenal of complementary DNA repair pathways has been selected, which together are capable of detecting and removing virtually any type of DNA damage (Giglia-Mari et al., 2011; Hoeijmakers, 2001). Furthermore, cells can postpone or halt DNA duplication and chromosome segregation. This provides a window of opportunity to erase the damage before DNA replication converts it into permanent mutations or irreversible chromosomal aberrations (Toettcher, 2013). If the DNA damage is sufficiently extensive, cells can resort to a last-ditch defense process known as apoptosis, or programmed cell death. To prevent cancer from developing through damage-induced mutations in important growth control genes, cells commit “suicide” and sacrifice themselves with the biological goal of protecting the whole organism (Wang, 2001). However, this represents a secondary response that is only deployed as the last resort in overcoming DNA damage. The primary process to prevent or counteract the erosion of genes is DNA repair, a set of mechanisms that identify and correct damage in the DNA sequence. Because there are so many types of damage, it's just not feasible for a single mechanism to handle them all. So, each repair system has its own set of tasks, a specific spectrum of damage that it can detect and repair, while other repair systems handle different types of damage. However, the entire network of repair mechanisms is able to deal with almost any conceivable DNA damage (Giglia-Mari et al., 2011; Hoeijmakers, 2001; Huang & Zhou, 2021).
DNA Damage Response
DNA Damage Response (DDR) refers to any mechanism that recognizes and corrects DNA damage, including cell cycle checkpoints, damage tolerance, which promotes the bypassing of small DNA lesions encountered by polymerases during replication to prevent it from stopping; and also DNA repair mechanisms (Bi, 2015; Giglia-Mari et al., 2011). While some types of DNA damage repair quickly, complex DNA damage takes longer to repair. In the latter scenario, signaling pathways are activated to halt the cell cycle and allow time for repair (Vítor et al., 2020). The type of DNA damage is an essential component in DDR, so the signaling pathway triggered for repair is highly reliant on the damaging agent and the lesion produced (Alhmoud et al., 2020). DNA repair is generally classified into four different types: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR) and double-strand break repair (DBR) (Thomas Helleday et al., 2008; Luo et al., 2019; Sancar et al., 2004).
BER, NER, and MMR are all DNA excision repair processes that employ a similar approach entailing lesion detection and excision followed by resynthesis using error-free DNA polymerases, a process referred to as "cut-and-patch" repair (Hoeijmakers, 2001). Lesions can be altered bases, as recognized by BER and NER, or normal but mismatched bases, as MMR recognizes. During replication, errors that escape proofreading become substrates for MMR and are fixed shortly after replication (Kunkel, 2009; McCulloch & Kunkel, 2008). BER, on the other hand, corrects non-bulky small DNA lesions - that do not alter the helical structure of DNA - but are highly mutagenic and pose a significant threat to genomic fidelity and stability. Much of the damage is caused by spontaneous DNA degradation (Lindahl, 1993) but comparable damage can also be caused by environmental chemicals, radiation, or therapy with cytostatic drugs that block cell cycle progression, such as chemotherapy. Finally, NER is a versatile DNA repair system targeting a variety of bulky lesions. These include helix-distorting DNA lesions produced mainly by environmental mutagens (agents that irreversibly change the DNA sequence) such as ultraviolet (UV) radiation and chemical substances (Gillet & Schärer, 2006). Lesions, which are sites for excision repair processes, reside in one of the two DNA strands. In these instances, the healthy complementary strand serves as an accurate blueprint for repairing the damaged strand.
When the damage is too extensive, however, these repair processes are followed by cell cycle checkpoint activation, which halts cell cycle progression while providing cells with the opportunity to either complete repair or embark on apoptosis (Choi et al., 2015, 2016; Park et al., 2015). Ionizing radiation, for instance, induces DNA double-strand breaks (DSBs), which damage both strands of DNA. The original chromosome is cleaved into small fragments by DSBs, which has been shown to be extremely damaging to the cell. DSBs are endowed with the ability to induce chromosomal rearrangements, in which parts of one chromosome attach to another (Varga & Aplan, 2005). A handful of tumors are associated with such rearrangements. Genes are also severely disrupted in the process (Tsai & Lieber, 2010). These lesions are extremely cytotoxic and challenging to repair because the cell cannot simply replicate information from an undamaged strand. DSBs are fixed using two distinct pathways: homologous recombination repair (HRR) and non-homologous end-joining repair (NHEJ) (Cahill, 2006; T Helleday et al., 2007; Wyman & Kanaar, 2006).
NHEJ functions by “gluing” the two broken ends of the chromosome back together. This repair procedure is imprecise and often involves the deletion or insertion of a few nucleotides at the cleavage site. Because of this, NHEJ often results in mutation, yet this is still more advantageous than the loss of an entire chromosome (Alberts et al., 2003). In HRR, information from the homologous chromosome that matches the damaged one is employed to repair the break. In this procedure, the two homologous chromosomes (a pair of chromosomes with the same gene sequence, one inherited from the mother and one from the father) are brought together, and the unaltered region of the homologous is used as a template to repair the broken chromosome's ends (Kimball, 2022). HRR is more efficient than NHEJ and yields fewer mutations (Alberts et al., 2003). The cell cycle phase determines the allocation of duties among these repair mechanisms. Because HRR demands a homologous chromosome, it only functions in the S and G2 phases. Postmitotic cells and dividing cells in the G1 phase, on the other hand, require NHEJ to seal DSBs (Clancy, 2008). There are, however, numerous alternative repair pathways to those listed here. In order to keep the DNA in its optimal condition throughout life, each cell nucleus contains numerous repair proteins. Despite the fact that several of the repair systems eliminate lesions in a cut-and-patch response, they are orchestrated towards different mutagens and are mechanistically separate, requiring unique factors and effector molecules.
When Damage Turns Into Disease
Human genetic abnormalities illustrate the importance of proofreading and repair mechanisms. Hereditary malignancies, often known as hereditary cancer syndrome, are connected to mutations in genes that produce proofreading and repair proteins. Certain cancer patterns can be observed within families in such circumstances, such as relatives with a higher than average likelihood of developing a particular type of cancer at a young age, close relatives with a common kind of cancer, or multiple types of cancer affecting the same individual (Steinke et al., 2013). Lynch syndrome, also known as Hereditary Nonpolyposis Colon Cancer (HNPCC), is an autosomal dominant genetic disease, meaning that the transmission of a faulty gene from one parent to the offspring can cause disease. It is caused by a mutation in one of four genes involved in the production of DNA mismatch repair proteins (Pino et al., 2009). Because mismatched bases cannot be repaired, there is an increased rate of single nucleotide changes and genomic instability in the DNA of people with the condition. This increases the likelihood of developing various types of cancer at a younger age, particularly of the colon and endometrium, with Lynch syndrome accounting for 2% to 5% of all colorectal cancers (Lynch & de la Chapelle, 1999). The diagnosis of Lynch syndrome is carried out in two steps: If there is a suspicion (because a patient develops cancer at a particularly young age), the tumor tissue is examined for faulty mismatch repair. If such clues are true, a genetic mutation is looked for. Detection of a mutation confirms the diagnosis in the patient and allows for predictive testing of other family members (Steinke et al., 2013).
Another interesting situation is Xeroderma Pigmentosum (XP), a rare, inherited skin and neurological disorder characterized by extreme photosensitivity and a higher likelihood of UV-induced malignancies such as skin and mucosal cancers (Cleaver, 1968). Underlying this condition are mutations in the nucleotide excision repair system, and because this pathway is defective, most UV damage cannot be repaired. XP symptoms often manifest in infancy or early childhood, with half of affected children suffering severe sunburn after spending just a few minutes in the sun. Compared to healthy individuals, people with XP are more than 1,000 times more likely to develop skin cancer, with the first case of skin cancer often occurring before the age of 10 (DiGiovanna & Kraemer, 2012; Kleijer et al., 2008).
Conclusions
Like any other molecule, DNA is susceptible to a variety of chemical reactions. Because DNA stores a permanent copy of the cell's genome, changes in its structure are far more damaging than changes in other cell components, such as RNAs or proteins. Sources of DNA damage are constant, countless, and include endogenous and exogenous culprits, often resulting in irreversible changes in the DNA sequence. Mutations can arise when incorrect nucleotides are incorporated during DNA replication. In addition, other chemical modifications occur in DNA, either naturally or as a result of exposure to chemicals or radiation. Such DNA damage often results in a high mutation rate with adverse effects on cell development. While not all mutations are harmful, many can have far-reaching effects that put the entire organism at risk. As a result, cells have had to evolve efficient surveillance and repair mechanisms to efficiently fix damaged DNA. When DNA damage is severe, repair processes are accompanied by cell cycle checkpoint activation, allowing cells enough time to either complete repair or trigger apoptosis in an attempt to remove defective cells. Defects in DNA repair are at the foundation of a number of human hereditary disorders that affect a wide variety of organs and tissues but share a number of traits, most notably a proclivity to cancer. Furthermore, a number of cancer-related genes have been discovered to encode proteins essential for DNA repair. In addition, a number of cancer-related genes that code for proteins essential for DNA repair have been discovered. Unfortunately, due to their rarity, challenging genetics, and a multitude of clinical features, most diseases associated with DNA repair go undiagnosed.
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