DNA Damage Checkpoints
Abstract
In response to diverse genotoxic stresses, cells activate DNA damage checkpoint pathways to guard genomic integrity and promote the survival of the organism. Counting on DNA lesions and context, damaged cells with alarmed checkpoints are often eliminated by apoptosis or silenced by cellular senescence or can survive and resume cell cycle progression upon checkpoint termination. Over the past two years, a plethora of mechanistic studies have provided exciting insights into the biology and pathology of checkpoint initiation and signal propagation, and have revealed the varied ways during which the response can be terminated: through recovery, adaptation, or cancer-prone subversion. Such studies highlight the dynamic nature of those processes and help us to raised understand the molecular basis, spatiotemporal orchestration, and biological significance of the DNA damage response in normal and cancerous cells.
Introduction
The genome of eukaryotic cells is under constant attack. A wide diversity of lesions caused by environmental agents such as ultraviolet (UV) radiation in sunlight, ionizing radiation, and various genotoxic chemicals can arise in the DNA. Additionally, the genome is also threatened from within the cell. By-products of normal cellular metabolism, such as reactive oxygen species (ROS; i.e., superoxide anions, hydroxyl radicals, and hydrogen peroxide) derived from oxidative respiration and products of lipid peroxidation, can cause a spread of damage within the DNA. On the opposite hand, DNA-damaging agents like ionizing radiation, UV light (photodynamic therapy), and most chemotherapeutic agents are increasingly getting used to treat common disorders like arterial (re)stenosis (brachytherapy and drug-eluting stents) or cancer. Whereas DNA damage in terminally differentiated cells (such as muscle cells) gives rise to DNA damage repair to ensure the integrity of the transcribed genome, the induction of DNA damage in dividing cells leads to the activation of cell cycle checkpoints. These checkpoints halt the proliferating cell in its cell cycle progression so as to offer time to the DNA damage repair machinery to try to its work, thereby avoiding incorrect genetic information from being passed onto the progeny. Especially when mutations are accumulating, the chance of developing uncontrolled cell growth (oncogenesis) is substantial. a spread of lesions can occur in the DNA, including single- and double-strand breaks (DSBs), mismatches, and chemical adducts. Therefore, multiple repair pathways have evolved, each directed to a specific sort of lesion. Each pathway consists of various proteins forming a cascade so as to repair the damage as accurately as possible. Eventually, when the repair process fails, the cell cycle can be blocked permanently, resulting in a senescent state of the cell, or alternatively, apoptosis could also be induced. Both mechanisms prevent potentially harmful cells from dividing, ensuring that no mutations are inherited by a subsequent generation of cells.
Cell cycle and DNA damage checkpoints
The cell cycle in eukaryotic cells consists of 4 phases: gap or G1 phase, synthesis or S phase, G2, and mitosis M-phase, and one phase outside the cell cycle i.e., G0 phase. In the G1 phase, directly after mitosis, the cell increases in size and starts synthesizing RNA by the process known as, transcription and proteins by translation. In the subsequent S phase, DNA is replicated to produce the exact same copy of the genome for the next daughter cell. During the G2 phase, the cell will grow and make extra proteins to make sure that two viable daughter cells can be formed by it. RNA and protein syntheses that started within the G1 phase are continued during the S and G2 phases. Finally, the cell will enter the M phase. During this phase, the chromosomes are organized in such a fashion that two genetically identical daughter cells are produced, after which the entire cycle can start again. Cells also can stop dividing and remain in G0. They'll stay during this G0 phase for hours, days, weeks, or maybe for years before they begin dividing again or maybe stay in G0 permanently until the organism dies.
In budding yeast, the DNA damage checkpoints (DDC) are often activated in three phases of the cell cycle. DNA damage incurred in G1 phase activates a transient DNA damage response that temporarily delays S-phase onset, providing overtime for DNA repair before replication. Damage that arises during the S phase slows replication and triggers a coordinated effort between the replication fork and DNA repair machinery to resolve the damage. DNA lesions still present after completion of DNA replication activate the G2M checkpoint, which stalls cellular division until the damage is repaired. If repair is successful, the DDC is extinguished, and cells proceed through mitosis by a process known as recovery. However, sustained DNA damage doesn't prevent cellular division indefinitely, as both budding yeast and metazoans will deactivate the checkpoint and proceed through cell division without repairing DNA, a process called adaptation. DDC in yeast is sort of sensitive, with one DSB being capable of activating the DDC and evoking a strong G2M arrest.
In mammalian cells, a couple of DSBs (1–4 breaks) can mildly activate the DDC and end in only minor effects in cell cycle progression. Mammalian cells can often carry DNA lesions induced by replication stress to the subsequent G1 cycle, likely reflecting the upper DNA damage thresholds required for DDC activation and imposition of cell-cycle arrest in mammals. As a consequence, vertebrates are likely more susceptible to encountering unrepaired DNA within the following G1 phase and could be more reliant on G1 checkpoints for inducing cell-cycle arrest upon low levels of DNA damage.
DNA damage response
The cells have evolved a highly organized mechanism to combat the various DNA assaults, called as the DNA damage response (DDR). DNA damage responses are a regimented series of diverse steps activated by discrete specific pathways. During stress conditions, the prevalence of DNA lesions triggers an array of processes which include (1) sensing of DNA damage, repair or removal of DNA damage to revive the integrity of the genome; (2) cell cycle checkpoints activation which renders cell for damage repair; (3) activation of various cellular pathways of survival cell death for successful elimination of damaged deregulated cells. All these components involved for the successful commencement of DDR are aligned in a hierarchical manner and functionally categorized into sensors of injury, signal transducers, amplifiers, and finally effectors. The proteins involved within the DDR are critically regulated by many regulators like phosphatases, kinases, ubiquitin ligases, deubiquitinases, and other protein modifying enzymes that modulate the activity and levels of key proteins.
Recognition of DNA damage at the site, recruitment of the DNA repair machinery, and commencement of repair process
Cells have different biochemical pathways to revive altered genomic integrity and preserve cellular homeostasis. Cells have developed various sets of machinery for the detection and repair of varied sorts of DNA damage. The repair of a specific DNA lesion is carried out by a specific pathway thus, making the list of repair pathways long.
1. Mis-match repair pathway
MMR is liable for the recognition and repair of base–base matches and insertion-deletion loops (IDLs) which are caused by faulty DNA replication and homologous recombination. To preserve genome integrity, MMR must happen selectively on the newly synthesized DNA strand containing the error. MSH2-containing complexes recognize DNA lesions followed by the recruitment of MLH13 and PMS12 complexes, then the endonucleases PMS2 and MLH3 make an incision at the location of the DNA lesion. Upon the marking of the acceptable strand by incision, the exonuclease Exo1 generates a multi-nucleotide gap which is filled and ligated by DNA polymerase? and Ligase I. Significantly, MMR increases DNA replication fidelity by about 100-fold.
2. Base excision repair
Base excision repair (BER) fixes non-bulky DNA base damage, abasic sites, and DNA single-strand breaks throughout all stages of the cell cycle. BER occurs in five major steps: (i). recognition and excision of a damaged base, (ii) incision at the abasic site, (iii) replacement of the excised DNA nucleotide, (iv) processing of DNA ends, and, (v) sealing of the DNA nick. DNA glycosylases are liable for the recognition and hydrolysis of DNA lesions followed by DNA polymerase? and XRCC1- ligase III-mediated nucleotide replacement and DNA nick sealing. SSBs are detected by PARP1 which catalyzes the formation of poly-ADP-ribose (PAR) chains on itself and other proteins to facilitate the recruitment of specialized BER enzymes and DNA-repair factors, like XRCC1, DNA polymerase? Ligases I and III.
3. Nucleotide Excision Repair
NER mainly fixes “bulky” DNA alterations including ultraviolet (UV)-induced photoproducts, base adducts created by genotoxic agents, for example, cisplatin, reactive oxygen species (ROS)-induced base modifications. NER occurs in four main steps: (1) DNA-damage recognition, (2) incision on each side of the DNA lesion and removal of the damaged DNA fragment, (3) gap-filling DNA synthesis, and (4) ligation of open DNA ends. About 30 proteins function within the NER pathway, and defects in NER components are linked to human diseases, like xeroderma, Cockayne syndrome, etc. Often, the NER pathway are subdivided into two processes: (1) the global genome NER (GG-NER) functioning during a cell cycle–an independent manner to get rid of UV-induced photoproducts and other “bulky” lesions, and (2) transcription-coupled NER (TC-NER) recognizing RNA polymerase stalled at “bulky” DNA lesions. GGNER and TC-NER differ at the step of DNA recognition damage and share the remaining DNA-repair machinery.
4. DNA Double-Strand Break
DNA Double-Strand Breaks are severe lesions which will end in the acquisition of disease-promoting properties or premature necrobiosis when not repaired properly. To attenuate the impact of DSBs, mammalian cells utilize different DSB-repair pathways. The 2 major pathways are: more error-prone but fast DNA non-homologous end joining (NHEJ) and error-free but slow Homologous recombination repair (HRR).
Depending on the origin of DSBs, mammalian cells use different DSB-repair mechanisms. DSBs can arise during programmed DNA recombination or accidental DNA breakage upon the arrest or stalling of DNA replication and exposure to DNA-damaging agents, such as IR or topoisomerase poisons. These events cause DSBs of a particular nature which is recognized and processed differently. The regulated resection of DSBs represents a key step within the choice between NHEJ and HRR where the accumulation of 53BP1 at DSBs can block the resection and consequently RAD51 loading, hence influencing DSB-repair pathway choice. Thus, 53BP1 generally promotes NHEJ and restricts HRR. Specifically, 53BP1 recruitment of RIF1 promotes NHEJ, while by excluding 53BP1 from DSB sites, BRCA1 can promote HRR.
DNA Damage- induced checkpoints
DNA Double-Strand Breaks provoke DNA damage response (DDR) activation, which reversibly impedes cell cycle progress to permit time for DNA repair. Once the DNA is repaired successfully, the cell recommences the cell cycle by switching off the DDR. Cdks (Cyclin-dependent kinases) control transitions of the cell cycle as they depend on and necessitate cyclin binding for its activity and substrate selectivity. The DDR elicits cell cycle arrest in G1 or G2 phase or can retard S- phase replication but it doesn't directly impede the mitosis process. Numerous mechanisms act synergistically to successfully accomplish the DDR in each phase of the cell cycle. Precise and subsequent activation of various arms of the cell cycle in response to DDR are described below:
1. The Main DNA Damage Sensors in G1-phase, ATM and ATR
Ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to DSBs while ATR gets activated in SSBs which subsequently trigger their downstream-regulatory signaling pathways. ATM works adjacent to DSB and induces phosphorylation of histone variant H2AX at S139 on the whole megabase chromatin domain resulting in cH2AX which amplifies ATM activity and recruits DNA repair machinery to the DSB site. ATM-mediated activation of downstream Chk2 kinase contributes to DNA damage repair by disseminating it from the damage site. G1 phase requires ATM and Chk2 for stabilization of p53 which successively activates a plethora of transcription factors alongside p21 (also known as CDKN1A, cyclin-dependent kinase inhibitor protein) Once accumulated, P21 causes the inhibition of Cyclin-Cdks complex activity by subsequent binding which halts cell cycle progression. Loss of p21 or p53 causes a complete loss of G1 checkpoint. ATM also regulates the activation of p38 MAPK which plays a big role within the commencement of G1 checkpoint as it stabilizes the p21 encoding mRNA levels (a relatively slow process). These molecules altogether rapidly but transiently prevent cells from entering the S-phase as they promote degradation of cyclin-D and Cdc25 phosphatase resulting in the reversal of inhibitory phosphorylation of Cdk2.
2. Signaling Downstream of ATM and ATR in S-Phase
Once the cell progresses through G1 phase, DSBs can trigger completely different signaling responses. Broken DNA ends are joined by the activation of HR repair process which involves extensive 5’-3’ resection of broken strands to make 3’ overhangs. This orchestrated HR repair system takes place only sister chromatids are available in the S or G2-phase of the cell cycle Ataxia-telangiectasia and Rad3-related (ATR) kinase gets activated on SSB generation and switches ATM, Chk2 axis in G1 phase to ATR, Chk1 axis in S and G2 phase of the cell cycle. Cdk2 phosphorylates two key exonucleases CtIP (Also referred to as RBBP8) and Exo1which ensures SSB end resection occurs only after entry of cell into S-phase Cdk2 dependent phosphorylation-mediated activation of ATRIP (ATR-interacting protein at S224) and Chk1 (at the S286 and S301) restricts TR and Chk1 activation to S- and G2-phase. Proliferating cell nuclear antigen (PCNA) associated CRL4-Cdt2 ubiquitin ligase (cullin 4A RING E3 ubiquitin ligase complex containing cdt2) prevents the buildup of p21. As p21 is continuously degraded, Wee1 kinase gets expressed in S-phase and inhibits Cdk2 activity by its phosphorylation. Wee1 also plays a big role in Cdc25A degradation to prevent further Cdk activation .3. Regulation of mitotic CDKs, leading to G2 arrest
G2M checkpoint gets triggered by ATR and Chk1, which thwarts the progression of cells to mitosis phase with damaged DNA. Commencement of mitosis requires the activity of cyclin B- dependent kinase 1 (Cdk1) which is that the master mitotic kinase. Wee1 and Myt1 are two regulatory Cdk1 inhibitory kinases which mediate the phosphorylation on T14 and Y15 to inhibit the catalytic activity of Cdk1 during the S and G2 phases. Cdc25C phosphatase removes these phosphorylations at the G2M transition. The progression of cells with damaged DNA to mitosis is mediated by ATR which inhibits cyclin BCdk1 activation by stimulating the Wee1 and inhibiting Cdc25C via Chk1.
4. M phase DNA repair completes the unfinished business
Progression through mitosis is regulated by the sequential proteolytic degradation of mitotic proteins like Plk-1, cyclin B, and securin, which is mediated by the ubiquitin-proteasome pathway. Mitotic kinase Plk1 phosphorylates Chk2 at multiple sites to thwart its dimerization and activation. Plk1 also abrogates Chk1 activity by stimulating the degradation of Claspin (scaffold protein) which plays an important role within the formation of a bridge between ATR and Chk1 during DSB repair. Plk1 also phosphorylates 53BP1 to obstruct its DNA repair functioning]. Cdk1-induced phosphorylation of RNF8 (at S139) hinders the formation of K63- linked polyubiquitin at the DNA damage site. Additionally, phosphorylation of XRCC4 by Plk1 and Cdk1 halts the NHEJ repair pathway.
Checkpoint deactivation
With or without successful repair, the DNA Damage Checkpoints are eventually terminated by dedicated deactivation mechanisms. Checkpoint deactivation is crucial to permit cell-cycle progression and cell proliferation, and mutants that fail to properly deactivate the checkpoint are sensitive to DNA damage. Deactivation of the DDC after the successful repair is related to the method of recovery, whereas DDC deactivation upon persistent damage is mentioned as adaptation. At the extent of kinases and kinase targets, deactivation is achieved primarily by the action of phosphatases, including, but not limited to only, PP2C and PP4 phosphatases in budding yeast and PP2A phosphatases in mammals. These phosphatases remove activating phosphorylations from key checkpoint components, like downstream checkpoint kinases and ?-H2AX. Because a cell-cycle arrest is usually established via the downstream checkpoint kinases Chk1CHK1 and Rad53CHK2, deactivation of those kinases allows rapid termination of the pro-arrest signaling. The checkpoint adaptors, like budding yeast Rad9, offer another key point of regulation, as their degradation or dis-engagement from DNA lesions prevents new checkpoint kinases from becoming active to take care of the cell-cycle arrest.
Summary
Taken together, eukaryotic cells believe in diverse signal transduction mechanisms to safeguard their genomic integrity. On the one hand, the accurate copying of genetic information within the S phase must be including the precise and equal distribution of chromosomes between daughter cells in mitosis. On the opposite hand, mechanisms must be in situ to detect and repair a broad range of DNA lesions that occur on a daily basis. Therefore, eukaryotic cells utilize DNA lesion-specific DNA damage–repair pathways to get rid of unwanted alterations of genetic information. Significantly, the repair of DNA lesions is synchronized with cell-cycle progression by the DDR. In response to DNA damage, a eukaryotic cell can respond with a transient cell-cycle arrest to permit the repair of DNA damage, or plan to a permanent cell-cycle arrest in the sort of senescence, or initiate apoptosis just in case the DNA damage is beyond repair. Generally, the DDR protects the cells against the buildup of DNA lesions which if not removed can cause human diseases including cancer, premature aging, and others. Thus, future research into bettering our understanding of DNA-damage checkpoints and DNA-repair mechanisms in health and disease is extremely likely to significantly expand our diagnosis, prediction, and treatment options in diverse human diseases.
