The Experts below are selected from a list of 35286 Experts worldwide ranked by ideXlab platform
Rajendra Prasad - One of the best experts on this subject based on the ideXlab platform.
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damage sensor role of uv ddb during base Excision Repair
Nature Structural & Molecular Biology, 2019Co-Authors: Sunbok Jang, Namrata Kumar, Emily C Beckwitt, Muwen Kong, Elise Fouquerel, Vesna Rapicotrin, Rajendra PrasadAbstract:UV-DDB, a key protein in human global nucleotide Excision Repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base Excision Repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are Repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase β-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG Repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.
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rev1 is a base Excision Repair enzyme with 5 deoxyribose phosphate lyase activity
Nucleic Acids Research, 2016Co-Authors: Rajendra Prasad, Vladimir Poltoratsky, Samuel H. WilsonAbstract:: Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base Excision Repair-inducing agent, we questioned whether Rev1 could also be involved in base Excision Repair (BER). Here, we uncovered a weak 5'-deoxyribose phosphate (5'-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.
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rev1 is a base Excision Repair enzyme with 5 deoxyribose phosphate lyase activity
Nucleic Acids Research, 2016Co-Authors: Rajendra Prasad, Vladimir Poltoratsky, Samuel H. WilsonAbstract:: Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base Excision Repair-inducing agent, we questioned whether Rev1 could also be involved in base Excision Repair (BER). Here, we uncovered a weak 5'-deoxyribose phosphate (5'-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.
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role of polymerase β in complementing aprataxin deficiency during abasic site base Excision Repair
Nature Structural & Molecular Biology, 2014Co-Authors: Melike Caglayan, Vinod K Batra, Akira Sassa, Rajendra Prasad, Samuel H. WilsonAbstract:5'-adenylated DNA adducts generated during nucleotide Excision Repair (NER) are removed by aprataxin to permit DNA end ligation. Now, structural and kinetic analyses reveal that NER enzymes DNA polymerase β and FEN1 can also excise these adducts and thus provide a 'backup' Repair pathway for abasic sites.
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requirement of mammalian dna polymerase beta in base Excision Repair
Nature, 1996Co-Authors: Robert W. Sobol, Rajendra Prasad, Julie K Horton, Ralf Kuhn, Hua Gu, Rakesh K Singhal, Klaus Rajewsky, Samuel H. WilsonAbstract:Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA Repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-Excision Repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-Excision Repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-Excision Repair in vivo.
Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.
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genome wide analysis of human global and transcription coupled Excision Repair of uv damage at single nucleotide resolution
Genes & Development, 2015Co-Authors: Jinchuan Hu, Sheera Adar, Christopher P Selby, Jason D Lieb, Aziz SancarAbstract:We developed a method for genome-wide mapping of DNA Excision Repair named XR-seq (Excision Repair sequencing). Human nucleotide Excision Repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of Repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine–pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD Repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled Excision Repair or general Excision Repair isolated the contribution of each pathway to the overall Repair pattern and showed that transcription-coupled Repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled Repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the Repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting Repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA Repair in human cells.
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genome wide analysis of human global and transcription coupled Excision Repair of uv damage at single nucleotide resolution
Genes & Development, 2015Co-Authors: Sheera Adar, Christopher P Selby, Jason D Lieb, Aziz SancarAbstract:We developed a method for genome-wide mapping of DNA Excision Repair named XR-seq (Excision Repair sequencing). Human nucleotide Excision Repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of Repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD Repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled Excision Repair or general Excision Repair isolated the contribution of each pathway to the overall Repair pattern and showed that transcription-coupled Repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled Repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the Repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting Repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA Repair in human cells.
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coupling of human dna Excision Repair and the dna damage checkpoint in a defined in vitro system
Journal of Biological Chemistry, 2014Co-Authors: Laura A Lindseyboltz, Joyce T. Reardon, Michael G Kemp, Vanessa Derocco, Ravi R Iyer, Paul Modrich, Aziz SancarAbstract:DNA Repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide Excision Repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide Excision Repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the Excision gap generated by the Excision Repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.
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nucleotide Excision Repair in human cells fate of the excised oligonucleotide carrying dna damage in vivo
Journal of Biological Chemistry, 2013Co-Authors: Joyce T. Reardon, Michael G Kemp, Jun Hyuk Choi, Shobhan Gaddameedhi, Aziz SancarAbstract:Nucleotide Excision Repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified Excision Repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled Repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the Repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide Excision Repair in humans.
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Regulation of nucleotide Excision Repair activity by transcriptional and post-transcriptional control of the XPA protein
Nucleic acids research, 2010Co-Authors: T-h Kang, Joyce T. Reardon, Aziz SancarAbstract:The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide Excision Repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances Excision Repair activity. Conversely, downregulation of XPA by siRNA reduces Excision Repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall Repair rate. Collectively, the data reveal that XPA is a limiting factor in Excision Repair and that its level is coordinately regulated by the circadian clock, the ubiquitin–proteasome system and DNA damage.
Samuel H. Wilson - One of the best experts on this subject based on the ideXlab platform.
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rev1 is a base Excision Repair enzyme with 5 deoxyribose phosphate lyase activity
Nucleic Acids Research, 2016Co-Authors: Rajendra Prasad, Vladimir Poltoratsky, Samuel H. WilsonAbstract:: Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base Excision Repair-inducing agent, we questioned whether Rev1 could also be involved in base Excision Repair (BER). Here, we uncovered a weak 5'-deoxyribose phosphate (5'-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.
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rev1 is a base Excision Repair enzyme with 5 deoxyribose phosphate lyase activity
Nucleic Acids Research, 2016Co-Authors: Rajendra Prasad, Vladimir Poltoratsky, Samuel H. WilsonAbstract:: Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base Excision Repair-inducing agent, we questioned whether Rev1 could also be involved in base Excision Repair (BER). Here, we uncovered a weak 5'-deoxyribose phosphate (5'-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.
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role of polymerase β in complementing aprataxin deficiency during abasic site base Excision Repair
Nature Structural & Molecular Biology, 2014Co-Authors: Melike Caglayan, Vinod K Batra, Akira Sassa, Rajendra Prasad, Samuel H. WilsonAbstract:5'-adenylated DNA adducts generated during nucleotide Excision Repair (NER) are removed by aprataxin to permit DNA end ligation. Now, structural and kinetic analyses reveal that NER enzymes DNA polymerase β and FEN1 can also excise these adducts and thus provide a 'backup' Repair pathway for abasic sites.
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requirement of mammalian dna polymerase beta in base Excision Repair
Nature, 1996Co-Authors: Robert W. Sobol, Rajendra Prasad, Julie K Horton, Ralf Kuhn, Hua Gu, Rakesh K Singhal, Klaus Rajewsky, Samuel H. WilsonAbstract:Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA Repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-Excision Repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-Excision Repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-Excision Repair in vivo.
Bo Tang - One of the best experts on this subject based on the ideXlab platform.
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Excision Repair initiated enzyme assisted bicyclic cascade signal amplification for ultrasensitive detection of uracil dna glycosylase
Analytical Chemistry, 2017Co-Authors: Lijuan Wang, Qianyi Zhang, Bo TangAbstract:Uracil-DNA glycosylase (UDG) is an important base Excision Repair (BER) enzyme responsible for the Repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using Excision Repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-Excision Repair, (2) Excision Repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA subs...
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Excision Repair initiated enzyme assisted bicyclic cascade signal amplification for ultrasensitive detection of uracil dna glycosylase
Analytical Chemistry, 2017Co-Authors: Lijuan Wang, Qianyi Zhang, Bo Tang, Ming Ren, Chun-yang ZhangAbstract:Uracil-DNA glycosylase (UDG) is an important base Excision Repair (BER) enzyme responsible for the Repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using Excision Repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-Excision Repair, (2) Excision Repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA Repair enzyme-related biomedical research and clinical diagnosis.
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Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase
2017Co-Authors: Lijuan Wang, Qianyi Zhang, Bo Tang, Ming Ren, Chun-yang ZhangAbstract:Uracil-DNA glycosylase (UDG) is an important base Excision Repair (BER) enzyme responsible for the Repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using Excision Repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-Excision Repair, (2) Excision Repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA Repair enzyme-related biomedical research and clinical diagnosis
Lijuan Wang - One of the best experts on this subject based on the ideXlab platform.
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Excision Repair initiated enzyme assisted bicyclic cascade signal amplification for ultrasensitive detection of uracil dna glycosylase
Analytical Chemistry, 2017Co-Authors: Lijuan Wang, Qianyi Zhang, Bo TangAbstract:Uracil-DNA glycosylase (UDG) is an important base Excision Repair (BER) enzyme responsible for the Repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using Excision Repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-Excision Repair, (2) Excision Repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA subs...
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Excision Repair initiated enzyme assisted bicyclic cascade signal amplification for ultrasensitive detection of uracil dna glycosylase
Analytical Chemistry, 2017Co-Authors: Lijuan Wang, Qianyi Zhang, Bo Tang, Ming Ren, Chun-yang ZhangAbstract:Uracil-DNA glycosylase (UDG) is an important base Excision Repair (BER) enzyme responsible for the Repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using Excision Repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-Excision Repair, (2) Excision Repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA Repair enzyme-related biomedical research and clinical diagnosis.
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Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase
2017Co-Authors: Lijuan Wang, Qianyi Zhang, Bo Tang, Ming Ren, Chun-yang ZhangAbstract:Uracil-DNA glycosylase (UDG) is an important base Excision Repair (BER) enzyme responsible for the Repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using Excision Repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-Excision Repair, (2) Excision Repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA Repair enzyme-related biomedical research and clinical diagnosis
