|Year : 2017 | Volume
| Issue : 2 | Page : 157-169
Regulation of various DNA repair pathways by E3 ubiquitin ligases
Chandramouli Natarajan1, Kenichi Takeda2
1 Riconpharma LLC, Denville, NJ, USA
2 Department of Multidisciplinary Internal Medicine, Division of Medical Oncology, Faculty of Medicine, Tottori University, Yonago, Japan
|Date of Web Publication||23-Jun-2017|
Riconpharma LLC, Denville, NJ
Source of Support: None, Conflict of Interest: None
DNA repair is the most important mechanism to maintain the normal cellular homeostasis. Owing to its complicated network, series of posttranslation modifications is required for proper function of the DNA repair proteins. One of such important posttranslation modifications is ubiquitination (attachment of ubiquitin). E3 ubiquitin ligases (UBLs) are a group of proteins that transfer ubiquitin from E2 conjugating enzymes to highly specific substrates such as DNA repair proteins. In this review, we have updated the role of different E3 UBL and how it regulates different DNA repair pathways.
Keywords: Base excision repair, DNA damage and repair, DNA damage tolerance, double-strand break repair, E3 ubiquitin ligases, nuclear excision repair
|How to cite this article:|
Natarajan C, Takeda K. Regulation of various DNA repair pathways by E3 ubiquitin ligases. J Can Res Ther 2017;13:157-69
| > Introduction|| |
Function of a protein can be greatly expanded, altered, and regulated by posttranslational modifications (PTMs). Modifications shall be in the form of attachment of small molecules such as phosphate, methyl, or acetyl groups. In this context, ubiquitin, a 76 residue protein, is also found to be covalently attached to other proteins and functionally alters them. Although initial function of ubiquitin-mediated modification was thought only to target the attached protein for proteasome-mediated degradation, later it was found that ubiquitin can also modify the targeted protein's location, conformation, stability, or activity.,,
Ubiquitin is synthesized as an inactive protein which is processed at its C-terminal to expose the glycine carboxylate – carboxyl group of this glycine is the site of attachment to substrates. Lysine side chains are the most common target sites within substrate proteins, resulting in an amide (or isopeptide) bond between the ubiquitin ligases (UBLs) and substrate. Ubiquitin also contains seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) and the amino group of the N-terminal Met which can also serve as substrates for ubiquitination, thus enabling the generation of different inter-ubiquitin linkage types. Although lysine is the most frequent substrate site for ubiquitin attachment, conjugation at different residues has also been reported.
The activation and attachment of ubiquitin involves three steps. In the first step, ubiquitin activating enzyme (E1) activates the COOH terminus of ubiquitin in an ATP-dependent manner and links through a thioester bond, to an active-site cysteine in the E1. In the second step, ubiquitin is transferred from the E1 to an active-site cysteine in ubiquitin-conjugating enzymes (E2). In the last step, UBL (E3) covalently links the ubiquitin to lysine residue of the target protein or to another ubiquitin molecule that has already been linked to the target protein by isopeptide bond.
There are two main types of E3 for ubiquitin (1) the RING class and (2) the HECT class: In RING class, the E3 binds to the ubiquitin thioester-linked E2 and substrate protein simultaneously and position the substrate lysine nucleophile in proximity to the reactive E2-ubiquitin thioester bond, facilitating transfer of the ubiquitin. In HECT class, the ubiquitin is first transferred from the E2 to an active site cysteine in the conserved HECT domain of the E3. The thioester-linked ubiquitin is then transferred to substrate. In humans, hitherto, two ubiquitin E1s, 38 ubiquitin E2s and more than 600 ubiquitin E3s, have been reported.
The fate of ubiquitin-modified proteins depends on the number and type of ubiquitin modifications., For proteasome degradation, at least four ubiquitins must be tagged to the protein that needs to be degraded. However, not all the polyubiquitin chain formation leads to proteolysis, and depending on the chain linkage, polyubiquitylated proteins play a role in recruiting proteins to specific sites, activating kinases and also act as a scaffold for diverse signaling processes., Mono-ubiquitinated proteins will undergo conformational change that leads to change in its interaction, activity, and localization.,,
The E3 ligases are considered to be the most important components of the ubiquitin conjugation machinery, as they bind directly to their target proteins and have substrate specificity. Hence, in this review, we discuss various DNA damage repair pathways and mechanisms of E3 UBL that are involved in mediating it.
| > DNA Repair|| |
The integrity of human genomic DNA is constantly challenged by intrinsic cellular metabolites, and exogenous DNA damaging agents. Even cells that are irradiated or exposed to chemotherapeutic agents induced DNA damage on the normal untreated cells.,,, It is estimated that each of the ~1013 cells within the human body incurs tens of thousands of DNA-damaging events per day. Different types of damages are repaired by different types of organized repair pathways. The DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). Ultraviolet (UV)-induced DNA lesions, and other bulky DNA adducts are repaired by nucleotide excision repair (NER), base lesions are repaired by base excision repair (BER) and DNA crosslinks are repaired by Fanconi anemia (FA) pathway. These DNA damages are recognized by sensor proteins which elicit cascades of PTMs including ubiquitination that regulates the repair.
| > Double-Strand Break Repair|| |
It has been estimated that approximately ten DSBs are induced per cell every day, and it is most lethal type of damage as it can induce cell death and genomic instability. DSBs are induced by exogenous agents such as ionizing radiation (IR), chemical exposure, whereas indigenous physiologic DSBs are induced by inadvertent action of nuclear enzymes, formation of reactive oxygen species by oxidative metabolism, V(D)J recombination in early lymphocytes of the vertebrate immune system and immunoglobulin gene class switch recombination (CSR).,,,, Another major cause of DSB includes replication across a nick, giving rise to chromatid breaks during S phase. DSBs are repaired by two major mechanisms: NHEJ and HR. The two pathways differ in their fidelity and template requirements. NHEJ modifies the broken DNA ends and ligates them together with little or no homology, generating deletions or insertions. In contrast, HR uses an undamaged DNA template on the sister chromatid or homologous chromosome to repair the break, leading to the reconstitution of the original sequence.
DNA damage activates the phosphatidylinositol 3-kinase-related kinases, especially ataxia telangiectasia mutated (ATM) for DNA DSB to phosphorylate H2AX at its C-terminus on serine 139 [Figure 1]. Phosphorylated H2AX (γH2AX) formation occurs within minutes after damage and extends up to a megabase from the site of the break in mammalian cells, providing a platform for subsequent DNA repair protein recruitment and amplification of repair. The γH2AX initiated by ATM gives a direct-binding platform for the mediator of DNA damage checkpoint protein 1 (MDC1) scaffold protein. The γH2AX-MDC1 interaction not only serves to recruit MDC1 to DSB sites but also protects γH2AX from protein phosphatases, such as PP2A or PP4, and prevents its removal from chromatin. MDC1 undergoes constitutive phosphorylation by Casein kinase 2 and DNA damage-induced phosphorylation by ATM, providing docking sites for RNF8 UBL.
|Figure 1: Role of E3 ubiquitin ligase RNF8 and RNF168 in double-strand break repair. MRN complex which sense double-strand break recruits ATM, H2AX, mediator of DNA damage checkpoint protein 1 and RNF8. (A) During homologous recombination, RNF8 monoubiquitinates gH2AX and RNF168 (E3) polyubiquitinates the monoubiquitinated gH2AX which is recognized by receptor-associated protein 80-Abraxas-BRCA1-BARD1 complex, which facilitates the double-strand break repair by homologous recombination. (B) Methylated histone H4 on K20 (H4K20me2) which is masked by polycomb protein L3MBTL1 and by Jumonji domain-containing protein 2A is ubiquitinated by both RNF8 and RNF168 and expose the methylated region on H4, which is immediately recognized and bound by 53BP1 and facilitates nonhomologous end joining|
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RNF8 is an E3 UBL which rapidly accumulates into IR-induced foci and co-localizes with γH2AX. RNF8 focus formation depends on MDC1 and γH2AX, but it is independent of NBS1, 53BP1, or BRCA1. The RNF8 forkhead-associated domain, specifically recognizes ATM-phosphorylated Thr-Gln-X-Phe (TQXF) motifs at the N-terminus of MDC1 on the one hand, and a unique ATM phosphorylation site in the extreme C-terminus of HERC2 (HECT domain E3 UBL) on the other hand.
The inherent ability of RNF8 is to associate with different E2 enzyme, this nature of RNF8 is altered during HERC2 association. HERC2 interaction causes RNF8 to specifically use UBC13 as E2 to form the nonproteolytic K63-linked polyubiquitin chains on histones at sites of DNA damage. Instead of directly ubiquitylating histones at sites of DNA damage, HERC2 assists RNF8 by modulating its preferred choice of cognate E2 ubiquitin-conjugating enzyme. In the absence of HERC2, this preference shifts toward other E2's possibly required for other functions of RNF8 in the cell.
The MDC1-RNF8-HERC2-UBC13 complex constitutes a UBL complex that is assembled on chromatin surrounding DSB's to initiate polyubiquitylation of γH2AX. Thus, instead of inducing the bulk of DSB-associated γH2AX polyubiquitylation, the RNF8-HERC2 complex appears to serve a priming role in this process, paving the way for a downstream E3 ligase, RNF168, which unlike RNF8 displays high activity toward H2A-type histone substrates.,
RNF168 is an E3 ligase that accumulates at sites of DNA damage within minutes following the induction of DSBs. Mutations of RNF168 are associated with radiosensitivity immunodeficiency dysmorphic features and learning difficulties (RIDDLE) syndrome, which is characterized by cellular defects in repairing DSBs. Cells derived from RIDDLE patients fail to recruit 53BP1 and BRCA1 at irradiation-induced DNA damage sites, which can be complemented by exogenous RNF168 expression.
RNF168 foci formation depends on RNF8 and MDC1 but not on BRCA1 or 53BP1. Further, it is also documented that RNF168 depletion does not affect the recruitment of MDC1 and RNF8 whereas it affects the localization of BRCA1 and 53BP1 to the sites of DNA damage. The observation was further substantiated with time lapse microscopy, where it was shown that RNF8 accumulates first at the site of DSBs, followed by RNF168 and with marked delay, by BRCA1. Together, these data indicate that RNF168 operates downstream of MDC1 and RNF8 to promote the re-localization of BRCA1 and 53BP1.
After its accumulation at the DNA damage site, RNF168 recognizes monoubiquitinated γH2AX at DSBs synthesized by RNF8 through its motif interacting with ubiquitin domains which represent an inverted ubiquitin-interaction motif. Like RNF8, RNF168 employs UBC13 as an E2 partner, and catalyzes K63-linked histone polyubiquitination at the DSB flanking chromatin primed by RNF8, to the levels sufficient to promote the ubiquitylation-dependent recruitment (UDR) of downstream factors such as 53BP1 and BRCA1., Thus, RNF8 plays a crucial role in the pathway by marking sites of DNA damage for RNF168 accrual and setting the stage for this highly active E3 ligase.
In contrast, recent study shows that RNF8 is inactive toward nucleosomal H2AX, whereas RNF168 catalyzes the monoubiquitination of the H2AX specifically on K13–15. Thus, recruitment of RNF168 in this model is still dependent on RNF8 but does not involve ubiquitylation of nucleosomal H2AX as the priming step, which suggests that that RNF8 only acts as a docking site for RNF168.
However, in both pathways, the central role played by RNF8/RNF168 is to polyubiquitinate H2AX which is necessary to orchestrate the DDR and repair pathways by recruiting BRCA1 and 53BP1. As we described earlier, DSBs are repaired by two major mechanisms: HR and NHEJ. 53BP1 and BRCA1 have reciprocal roles in DSB repair: BRCA1 is required for efficient HR, while 53BP1 promotes NHEJ.,
BRCA1 is a tumor suppressor protein which plays a major role in maintenance of genomic integrity. BRCA1 interacts directly or indirectly with numerous molecules, including tumor suppressors, oncogenes, DNA damage repair proteins, cell cycle regulators, transcriptional activators, and repressors., Its role in DDR is important as it was found to regulate checkpoint control, as well as DSB repair by HR.
BRCA1 encodes 1863 amino acid residues that contains a tandem BRCA1 carboxy-terminal (BRCT) domain in its C-terminal region and a RING finger domain in its N-terminal that specifically interacts with the structurally related RING domain protein BARD1 (BRCA1 associated ring domain protein 1), receptor-associated protein 80 (RAP80)-Abraxas-BRCA1 complex is recognized by K63-linked ubiquitin conjugates RNF8/RNF168 and recruits BRCA1 to the DNA damage site.
BRCA1 and BARD1 heterodimerize to form a functional E3 UBL, which in complex with E2 UBCH5C, promotes the formation of K6-linked ubiquitin chains., Even though cancer-predisposing mutations were found in both BRCT and RING domains, recent results showed that BRCT phospho-protein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.
The substrates for BRCA1 E3 ligase activity has not been revealed much, but two potential substrates, C-terminal-binding protein interacting protein (CtIP) and nucleoplasmin 1 (NPM1) that are involved in HR have been demonstrated.
C-terminal-binding protein interacting protein
CtIP is an important regulator of DNA end-resection, a process necessary for HR. CtIP recruited by MRN is phosphorylated by ATM at serine 327, then interacts with BRCA1, and ubiquitinated by BRCA1 and UBC13. Ubiquitinated CtIP promotes the loading of replication protein A (RPA) onto single-stranded regions, to protect single-stranded DNA (ssDNA) from nucleases and prevent hairpin formation in ssDNA that would interfere with DNA processing until the ssDNA is coated with RAD51. RAD51 then displaces RPA with the help of RAD52 which facilitates strand invasion at the homologous region of the sister chromatid. RING mutations in BRCA1 that disrupt E3 ligase activity or CtIP deficiency failed to support CHK1 phosphorylation via ATR, resulting in a defective IR-induced G2 checkpoint.
NPM is a protein found to be involved in ribosomal biogenesis, anti-apoptotic acitivity, centrosome duplication, DNA replication, recombination, transcription and repair.,, It was found that upon DNA damage, NPM1 translocates from the nucleolus to the nucleoplasm and binds to chromatin. It is possible that NPM1 could function as a histone chaperone under, or following, repair of DNA strand breaks. NPM1 is recruited to DSBs in a manner dependent on RNF8/RNF168-mediated ubiquitination. BRCA1 E3 ligase which is also recruited to DSB in a similar manner was found to ubiquitinate NPM1. The ubiquitination of NPM1 by BRCA1 does not cause its degradation instead; ubiquitination stabilizes NPM1 protein and may alter its histone chaperone activity, redistribution of NPM1 to the spindle poles. Recent study showed that APE1, a core enzyme in BER of DNA lesions interacts with NPM1 and its activity is stimulated by NPM1. However, the role of ubiquitinated NPM1 in DNA repair is yet to be revealed.
Apart from ubiquitination, DNA repair is also mediated by BRCA1-BARD1 complex based on its BRCT dependent interaction. BRCA1-BARD1 forms protein complexes with three different phosphorylated proteins such as Abraxas, BACH1 (FANCJ or BRIP1) and PALB2 and it has been proposed that these proteins may serve as adaptor proteins.
The localization of BRCA1 to damage-induced foci occurs in a BRCT dependent manner, but is independent of the CtIP and BRIP1., Studies showed following its phosphorylation on serine 406, Abraxas interacts specifically with the BRCA1 BRCT domain. Abraxas, a coiled-coil domain-containing protein 98, binds directly to BRCA1 at the same location and competes with BACHI and CtIP. The specific function of Abraxas is to recruit the RAP80 and bridges the interaction between RAP80 and BRCA1 BRCT domain. Like BRCA1, RAP80 accumulates into IR-induced foci in an MDC1 and γH2AX-dependent manner., This complex is involved in G2-M checkpoint control and ensures that entry into mitosis is transiently inhibited to avoid aberrant chromosome segregation.
BACH1/BRIP1 or FANCJ
BACHI is basically a helicase, and it is an important participant in DNA repair by HR, especially in response to cross-linking agents.,, BACHI mutations are associated with increased risk of breast cancer. Following DNA damage, BACH1 becomes phosphorylated by ATM. Phosphorylated BACH1 (serine 990) is required for its attachment with BRCA1 and this binding result in the prevention of RAD51 displacement, an important event for HR.
Recent results have also yielded important insights into the regulation and function of the products of BRCA1 and PALB2 (FANCN), tumor suppressor genes in cellular responses to DNA damage. Results suggest that BRCA1-dependent localization of PALB2 may link DNA repair by HR into a pathway with the MDC1-RNF8-RAP80-Abraxas signaling cascade. Thus, in this manner, PALB2-dependent BRCA2 initiates HR-mediated DNA repair by recruiting RAD51 to sites of DNA damage and facilitating its assimilation onto ssDNA.
53BP1 (also called TP53BP1) is a large protein of 1972 amino acids that has BRCT repeats, tandem Tudor domains, and 28 amino-terminal Ser/Thr-Gln (S/T-Q) sites, which are phosphorylated, at least in part, by the ATM kinase.
The recruitment of 53BP1 to the DSB was shown to occur via recognition of mono or dimethylated histone H4 on K20 (H4K20me2) by its tandem Tudor domain., The Tudor domain of 53BP1 recognizes the dimethylated H4K20 upon DNA damage, which is otherwise masked by the polycomb protein L3MBTL1 and by the demethylase Jumonji domain-containing protein 2A (JMJD2A; also known as KDM4A). L3MBTL1 were found to be degraded upon DNA damage by VCP (molecular chaperone valosin-containing protein) which accumulates in an RNF8-dependent manner. Whereas, JMJD2A was polyubiquitinated and degraded in response to DNA damage by both RNF8 and RNF168. Hence, the activation of the RNF8/RNF168 pathway induces the ubiquitin-dependent removal of these proteins from damaged chromatin and exposes the H4K20me2 mark to enable increased 53BP1 binding.
A recent study shows that 53BP1 not only binds to dimethylated H4K20 but also engages with ubiquitylated form of H2A: H2AK15U. The study proposes that 53BP1 forms a dimer and binds to the H2A ubiquitinated on Lys-15 (H2AK15ub) a product of RNF168 action using UDR motif. Engagement of H4K20me2 by the Tudor domain of 53BP1 positions the UDR motif of 53BP1 in the correct orientation to contact the epitope formed by H2AK15ub.
In contrast, recent study revealed that RNF168 associates with 53BP1 independently of the γH2AX-MDC1-RNF8 signaling axis and ubiquitylates at lysine 1268 of 53BP1 before its localization to DSB sites, and this K63 but not K48-linked ubiquitylation is important for the initial recruitment of 53BP1 to DSB sites and its function in NHEJ and activation of checkpoints.
Even though 53BP1 is not an E3 ubiqutin ligase, it is important to discuss its role because of its importance in NHEJ based DSB repair. One of the important functions of 53BP1 is that it antagonizes the resection of DSBs in G1, preserving DSB ends and thereby favoring repair by NHEJ over HR. 53BP1 contributes to NHEJ in CSR, long range V(D) J recombination, the fusion of dysfunctional telomeres deprived of telomeric repeat-binding factor 2 protection, and maintaining genomic stability. BRCT domain of 53BP1 also interacts with damage responsive proteins like RIF1 and PTIP. RIFI which accumulates in a 53BP1 dependent manner block DSB resection in G1. PTIP is a transcription regulator that contributes to the fusion of dysfunctional telomeres and also involves in DSB end-resection, similarly to 53BP1 and RIF1.
| > Nucleotide Excision Repair|| |
Bulky DNA adducts that are induced by external agents such as UV and chemicals are repaired using NER., It is also estimated that out of 3 × 109 bases in the genome, intracellular metabolism induces spontaneous base damage in 25,000 bases per human genome per cell per day that cause alterations in the chemistry and structure of the DNA duplex. UV radiation induces two main types of lesions, cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (PPs). Both of these lesions alters DNA structure and thereby inhibits DNA transcription and replication. NER is initiated by recognizing the DNA damage and assembling its preincision complex over it, then the damaged strand was excised from the DNA backbone and new strand were synthesized and finally the nicks were sealed with ligases. NER also plays a partially significant role in repairing cross-link formation.
NER recognizes and removes helical distortions in two modes: Global genome (GG)-NER which preferentially removes lesions from total genomic DNA (coding parts of the genome and nontranscribed strands of active genes). Transcription-coupled (TC)-NER is restricted to removing lesions preferentially from the transcribed DNA strand of active genes using RNA polymerase II (RNAPII) as the sensing complex [Figure 2].
|Figure 2: Nucleotide excision repair is repaired by two main pathways called global genome-nucleotide excision repair and transcription coupled-nucleotide excision repair. (A) During DNA damage, CUL4 E3 ligase complex recruits and ubiquitinates xeroderma pigmentosum Group C and increases its sensitivity to recognize and bind the distorted DNA. Xeroderma pigmentosum Group C recruits further nucleotide excision repair core repair components to complete the repair of global genome-nucleotide excision repair. (B) In transcription coupled-nucleotide excision repair, RNA polymerase II recognizes the distorted DNA and recruits Cockayne syndrome Type B. Cockayne syndrome Type B then recruits CUL4 E3 ligase and core nucleotide excision repair proteins and also ubiquitinates Cockayne syndrome Type B. Ubiquitinated Cockayne syndrome Type B detaches from the distorted DNA which otherwise acts as physical block for the repair process and transcription|
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CUL4-DDB2 in global genome-nucleotide excision repair
In GG-NER, xeroderma pigmentosum Group C (XPC) complex is the lesion sensing component which consists of XPC, RAD23 and centrin 2. XPC as the DNA binding subunit is very inefficient at recognizing CPDs, whereas it can easily recognize PPs. To assist XPC in recognizing CPDs, DNA-damage binding (DDB) complex, comes into play. This E3 complex consists cullin 4A as a scaffold, an E2-binding subunit (RBX1/ROC1 or RBX2) on one side, adaptor subunit DDB1 on another side with DDB2/XPE - a target receptor that is capable of sensing even the little distorted DNA., Once the DDB complex recognizes the DNA damage, the CUL4 subunit gets neddylated and activates the E3 ligase activity of the complex. This activated E3 complex then recruits XPC complex to the DNA damaged site.
Further, with UBCH5 as E2, it ubiquitinates XPC and increases XPC's affinity for DNA. RAD23 which is associated with XPC protects it from ubiquitin-mediated proteasome degradation. Then the XPC binds to the intact strand opposite to damaged site and recruits transcription factor II human (TFIIH). The DDB2 and CUL4A were also found to self-ubiquitinate, which decreases its affinity for DNA and eventually triggers its degradation. Thus, the complex hands-over the damaged site to XPC by increasing its affinity at the same time reducing its own., After DNA damage recognition, both GG and TC-NER were found to follow a common pathway in which the damaged DNA is un-winded by TFIIH complex with the help of helicases XPB and XPD. XPA binds to the site of DNA damage, while RPA binds to the undamaged DNA and allows the binding of endonucleases XPG (3') and XPF-ERCC1 (5') to excise the damaged nucleotides. New strands are synthesized with the help of DNA polymerase and proliferating cell nuclear antigen (PCNA). Finally, the nicks were sealed by DNA ligase to complete the repair process.
CUL4-CSA in transcription coupled-nucleotide excision repair
Unlike GG-NER where ubiquitination is necessary for the DNA damage sensing; in TC-NER the recognition is done without UBL, but the role of ubiquitin was found to be necessary for the resumption of transcription.
In TC-NER, RNAPII which stalls upon a DNA lesion recruits Cockayne syndrome Type B (CSB). CSB then recruits the E3 complex which consists cullin 4A as scaffold, DDB1 as adaptor subunit and CSA as the target receptor that senses distorted DNA. CSA recruits the core NER machinery  to complete the repair. After repair, the CSB complex may act as a physical block for RNAPII to resume transcription. During this time CSA complex polyubiquitinate and degrades CSB with the help of UBCH5 as an E2, resuming the transcription.
It has also been reported that RNAPII is rapidly ubiquitinated by Nedd4 during TCR-NER and speculating that its ubiquitination may result in detaching if from DNA and degraded by 26S proteasome, which might be a signal to recruit additional NER enzymes.
| > DNA Damage Tolerance|| |
The presence of bulky DNA adducts was actively repaired by NER. When the unrepaired bulky DNA adducts or gaps are encountered by replication fork in actively replicating DNA, it stalls DNA polymerases. Prolonged stalling of replication forks lead to fork collapse and ultimately genome instability. These types of damages were bypassed by DNA damage tolerance (DDT) pathway which promotes DNA replication, leaving the damage to be repaired at later time.,, DDT or postreplicative repair is regulated either by an error-prone translesion synthesis (TLS) or error-free recombination by template switching, based on the ubiquitin state of PCNA, [Figure 3]. PCNA forms a doughnut-shaped homotrimeric clamp which encircles DNA. PCNA serves as a processivity factor and recruits both replicative and specialized TLS polymerases during normal DNA replication and postreplicative DNA repair respectively.,,
|Figure 3: In actively replicating DNA, the damage is bypassed by DNA damage tolerance or postreplicative repair. Based on the ubiquitin status of proliferating cell nuclear antigen, DNA damage tolerance may result in error-prone translesion synthesis or error-free template switching. E3 ligase RAD18 and E2 RAD6 monoubiquitinates proliferating cell nuclear antigen and signals the bypass mechanism to undergo translesion synthesis. Whereas, E3 ligase SNF2 histone-linker PHD-finger RING-finger helicase/helicase-like transcription factor and E2 UBC13/MMS2 polyubiquitinates proliferating cell nuclear antigen and signals the bypass mechanism to undergo template switching|
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RAD18 in translesion synthesis
During replication stall, the ssDNA coated with RPA recruits the RAD18 (E3) and RAD6 (E2) UBL that monoubiquitinate PCNA at Lys-164.,, E2 enzyme RAD6 is intrinsically capable of catalyzing polyubiquitin chain formation. PCNA polyubiquitination is prevented by RAD18 which inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on RAD6. Monoubiquitinated PCNA was immediately recognized by UBD containing Y family (TLS) polymerases that lack high specificity for Watson-Crick base pairs. TLS polymerases can accommodate bulky DNA adducts in their active sites, which ultimately bypasses the lesion and synthesis DNA across the damaged site. After lesion bypass, PCNA is deubiquitinated which results in reverting of replicative polymerase in the place of TLS polymerase.
Apart from postreplication repair RAD18 was also found to have a role in the efficient repair of DSB by regulating FANCD2, meiotic DSB, and DSB tolerance during G1 by ubiquitinating 53BP1 at lysine 1268 and its retention at sites of DNA damage.
SNF2 histone-linker PHD-finger RING-finger helicase and helicase-like transcription factor in template switching
When TLS fails, E3 ligases - SNF2 histone-linker PHD-finger RING-finger helicase and helicase-like transcription factor (HLTF); a functional human homolog of yeast RAD5 in association with UBC13/MMS2 (E2) extends the monoubiquitination by linking the K63 linked polyubiquitination of PCNA., The mechanism of polyubiquitinated PCNA regulation by template switching is exactly not known. However, recent study showed that polyubiquitinated PCNA recruits the ZRANB3 translocase, also known as AH2, to sites of replication stress. ZRANB3 catalyzes the regression of DNA substrates that mimic stalled replication forks and disassembles D loop recombination intermediates and thereby maintains genomic stability and prevent inappropriate recombination that could take place during template switching events at sites of replication stress.,, Apart from polyubiquitinating PCNA, recent studies showed HLTF is also involved in a RAD51 independent D loop branch formation for template switching pathway in addition to its fork reversal activity.
| > Base Excision Repair|| |
Small DNA lesions that results from hydrolytic loss of DNA bases, oxidation, nonenzymatic methylations, alkylation or deamination, modifies the structure of DNA double helix.,, These modifications are recognized and repaired by the BER pathways otherwise leads to genomic instability. DNA glycosylases initiate BER by recognizing the damaged DNA base lesion and cleaves the N-glycosidic bond [Figure 4]. The resulted abasic site is processed by DNA AP endonuclease or a DNA AP lyase that cleaves the phosphodiester bond 5' to the AP site and removes it. DNA polymerase incorporates a new nucleotide and the nick is sealed by ligase.,,,
|Figure 4: DNA polymerase B levels were regularly maintained in base excision repair by ubiquitin mechanism. In normal cells, the excess DNA pol b is polyubiquitinated and degraded by E3 ligase Mule in association with E2 UBCH5C. In DNA-damaged cells, damaged induced ARF inhibits the ubiquitin activity of Mule, thus protecting the degradation of Pol b and increases it availability for repair|
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Mule and CHIP in base excision repair
Among the BER enzymes, DNA polymerase Pol b is the central component which fills the single nucleotide inside the gap.,, Steady state levels of Pol b should be maintained for an effective BER as its underexpression or overexpression leads to deficient repair or increased mutation respectively., The Pol b levels were maintained by two ubiquitin E3 ligases Mule and CHIP in association with E2 UBCH5C. Studies showed that in undamaged cells, the excess pol b is monoubiquitinated by Mule which in turn was recognized and polyubiquitinated by CHIP. Thus polyubiquitinated Pol b is degraded and its levels were kept in check until DNA damage., During DNA damage, the level of acute rheumatic fever protein is increased, which inhibits Mule activity and thus increases pol b level and its availability for the repair. This cycle is repeated constantly to adjust the repair capability of cells with the amount of DNA damage.,
| > Fanconi Anemia|| |
FA is a chromosomal instability disorder characterized by bone-marrow failure and susceptibility to acute myelogenous leukemia, squamous cell carcinoma of head and neck, hepatocellular carcinoma, congenital abnormalities and infertility., Germline mutations are identified in 19 genes that lead to FA because of their compromised function in DNA repair. FA proteins participate in the repair of extraordinarily deleterious lesions, inter-strand crosslinks, and in maintaining genomic stability during DNA replication [Figure 5].,, FA highly regulates the interstrand cross-link (ICL) by involving various major repair pathways like nuclease from NER, polymerases from TLS and HR to resolve DSB.
|Figure 5: Ubiquitination of Fanconi anemia proteins FANCD2 and FANCI is considered as an important step in repairing interstrand cross-link. Replication fork stalled upon detecting interstrand cross-link recruits Fanconi anemia core complex and accessory proteins. The E3 ligase FANCL containing core complex with E2 UBE2T ubiquitinates both FANCD2 and FANCI and localizes them into chromatin. The chromatin localized ubiquitinated D2 and I then recruits the nucleases from nucleotide excision repair, polymerases from translesion synthesis and recombination proteins from homologous recombination machinery to complete the interstrand cross-link repair|
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FANCM-FAAP23-MHF ½ complex recognizes the stalled replication fork structure and recruits the FA core complex to the ICL region., Among the 19, 8 FA proteins (FANCA/B/C/E/F/G/L/M) and accessory proteins (FAAP20, FAAP24 and FAAP100) form a multi-subunit ubiquitin E3 ligase complex or the FA core complex.
During crosslink detection, the core complex is phosphorylated by ATR and activates them. FANCL does the ubiquitin E3 ligase function among the core complex and UBE2T providing its services as an E2 ligase, both of them monoubiquitinate FANCD2 and FANCI at Lys-561 and 523 respectively and activates them., Monoubiquitination of FANCD2 and FANCI are considered as an important step as it activates the FANCI-FANCD2 complex which then coordinates the action of downstream repair factors. Once monoubiquitinated, these FANCD2 and FANCI complex localized into the chromatin, and interacts with the downstream components which consists of nucleases (XPF, MUS81 and SLX1),, HR proteins (BRCA2, BRIP1, PALB2 and RAD51C),,,,, and scaffold proteins (SLX4),, and completes the repair.
Apart from its function in ICL, the ubiquitinated FANCD2 was found to be important to deposit RAD51 onto the fork under replication stress and thereby activating HR. In the absence of FA pathway, error-prone NHEJ was found to be more predominant in repairing DNA DSB, indicating the importance of FA.,
Even though the function of RAD18 in regulating FA pathway is not clear, it has been reported that RAD18 is necessary for chromatin loading of FANCD2 in untreated cells, suggesting that RAD18 modifies proteins upstream of FANCD2 and FANCI monoubiquitination.
Reports have also shown that RAD18-mediated PCNA monoubiquitination is required for FA pathway activation via a mechanism involving recruitment of FANCL to chromatin and subsequent ID complex monoubiquitination. In contrast, PCNA-independent role of RAD18 in DSB repair has also previously been proposed which shows that RAD18 E3 UBL activity is essential for FA pathway activation and damage tolerance after CPT treatment.
| > Conclusion|| |
DNA repair is one of the well-regulated networks to overcome the damages that are experienced by cells in their lifetime, which otherwise leads to genomic instability. While deficient DNA repair results in genomic instability in normal cells, active DNA repair in cancer cells often leads to chemoresistance., One of the signaling mechanisms that actively regulates DNA repair in both normal and cancer cells is ubiquitination. Although all the functions of ubiquitin are not revealed, it is very important to note that ubiquitin has a major role in regulating the DNA damage repair network [Table 1]. By its proteolytic or nonproteolytic mechanism, ubiquitin regulates the DNA repair proteins by recruiting, stabilizing, degrading, altering cellular localization, and helps in linking with other proteins. The other two important components of this repair network which is not mentioned in this review but possess equal functional importance like ubiquitin are small ubiquitin-related modifier (SUMO) and deubiquitinating enzymes (DUBs).
SUMO-like ubiquitin can be attached to a protein and change its property, stability, and localization. SUMO is also found to regulate various DNA repair mechanisms and its failure also results in cancer formation. DUBs belong to superfamily of proteases that cleave ubiquitin from polypeptide substrates such as ubiquitin precursors, ubiquitin adducts, polyubiquitin chains, mono and polyubiquitylated proteins. DUBs regulating the processes of HR, NHEJ, NER, BER, TLS, FA, and mismatch repair have been identified. Aberrant expression of DUBs is also reported in human cancers.
The malfunction of ubiquitin's proteolytic and nonproteolytic role results in various cancer formations. Apart from cancer, the role of ubiquitin is also involved in differentiation, development, inflammation, cell cycle, viral infection, neural degeneration, etc. Novel findings of ubiquitin mechanism and relating it better with other PTMs such as phosphorylation and methylation will help us target these molecules for diagnostic and therapeutic approaches.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]