The Experts below are selected from a list of 492 Experts worldwide ranked by ideXlab platform
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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structure and mechanism of the 2 3 phosphatase component of the bacterial PNKP hen1 rna repair system
Nucleic Acids Research, 2013Co-Authors: Li Kai Wang, Paul Smith, Stewart ShumanAbstract:PNKP is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. PNKP is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn2+-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum PNKP with Mn2+ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn2+ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum PNKP phosphatase.
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mechanism of rna 2 3 cyclic phosphate end healing by t4 polynucleotide kinase phosphatase
Nucleic Acids Research, 2013Co-Authors: Ushati Das, Stewart ShumanAbstract:T4 polynucleotide kinase-phosphatase (PNKP) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the PNKP C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid-base catalyst Asp167. We report that PNKP also has RNA 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for PNKP phosphatase is an RNA 2',3'-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that PNKP reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type PNKP transforms RNA > p to RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + PNKP → RNA-(3'-phosphoaspartyl)-PNKP → RNA(3')p + PNKP → RNA(OH) + phosphoaspartyl-PNKP → P(i) + PNKP.
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structure and mechanism of the polynucleotide kinase component of the bacterial PNKP hen1 rna repair system
RNA, 2012Co-Authors: Li Kai Wang, Paul Smith, Ushati Das, Stewart ShumanAbstract:PNKP is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. PNKP is composed of three catalytic modules: an N-terminal polynucleotide 5'-kinase, a central 2',3' phosphatase, and a C-terminal ligase. Here we report the crystal structure of the kinase domain of Clostridium thermocellum PNKP bound to ATP•Mg²⁺ (substrate complex) and ADP•Mg²⁺ (product complex). The protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain. ATP is bound within a crescent-shaped groove formed by the P-loop (¹⁵GSSGSGKST²³) and an overlying helix-loop-helix "lid." The α and β phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides; the γ phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg²⁺ bridge the ATP β and γ phosphates. The P-loop serine (Ser22) is the sole enzymic constituent of the octahedral metal coordination complex. Structure-guided mutational analysis underscored the essential contributions of Lys21 and Ser22 in the ATP donor site and Asp38 and Arg41 in the phosphoacceptor site. Our studies suggest a catalytic mechanism whereby Asp38 (as general base) activates the polynucleotide 5'-OH for its nucleophilic attack on the γ phosphorus and Lys21 and Mg²⁺ stabilize the transition state.
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the adenylyltransferase domain of bacterial PNKP defines a unique rna ligase family
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Paul Smith, Pravin A Nair, Li Kai Wang, Stewart ShumanAbstract:PNKP is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from ten different phyla. To gain insight to the mechanism and evolution of this repair system, we determined the crystal structures of the ligase domain of Clostridium thermocellum PNKP in three functional states along the reaction pathway: apoenzyme, ligase•ATP substrate complex, and covalent ligase-AMP intermediate. The tertiary structure is composed of a classical ligase nucleotidyltransferase module that is embellished by a unique α-helical insert module and a unique C-terminal α-helical module. Structure-guided mutational analysis identified active site residues essential for ligase adenylylation. PNKP defines a new RNA ligase family with signature structural and functional properties.
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Mechanism of RNA 20,30-cyclic phosphate end healing by T4 polynucleotide kinase–phosphatase
2012Co-Authors: Ushati Das, Stewart ShumanAbstract:T4 polynucleotide kinase–phosphatase (PNKP) exemplifies a family of enzymes with 50-kinase and 30-phosphatase activities that function in nucleic acid repair. The polynucleotide 30-phosphatase reaction is executed by the PNKP C-terminal domain, which belongs to the DxDxT acyl-phosphatase superfamily. The 30-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid–base catalyst Asp167. We report that PNKP also has RNA 20-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for PNKP phosphatase is an RNA 20,30-cyclic phosphate end (RNA>p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that PNKP reactivity with RNA>p requires Asp165, but not Asp167. Whereas wild-type PNKP transforms RNA>p to RNAOH, mutant D167N converts RNA>p to RNA 30-phosphate, which it sequesters in the phosphatase active site. In support of the intermedi-acy of an RNA phosphomonoester, the reaction of mutant S211A with RNA>p results in transient ac-cumulation of RNAp en route to RNAOH. Our results suggest that healing of 20,30-cyclic phosphate ends is a four-step processive reaction: RNA>p+Pnk
Michael Weinfeld - One of the best experts on this subject based on the ideXlab platform.
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domain analysis of PNKP xrcc1 interactions influence of genetic variants of xrcc1
Journal of Biological Chemistry, 2019Co-Authors: Rajam S Mani, Ismail Abdou, Mesfin Fanta, Inbal Mermershtain, Michael J. Hendzel, J Mark N Glover, Michael WeinfeldAbstract:Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.
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nanoencapsulation of novel inhibitors of PNKP for selective sensitization to ionizing radiation and irinotecan and induction of synthetic lethality
Molecular Pharmaceutics, 2018Co-Authors: Zahra Shire, Timothy D R Morgan, Mohammad Reza Vakili, Dennis G. Hall, Afsaneh Lavasanifar, Michael WeinfeldAbstract:There is increasing interest in developing and applying DNA repair inhibitors in cancer treatment to augment the efficacy of radiation and conventional genotoxic chemotherapy. However, targeting the inhibitor is required to avoid reducing the repair capacity of normal tissue. The aim of this study was to develop nanodelivery systems for the encapsulation of novel imidopiperidine-based inhibitors of the DNA 3′-phosphatase activity of polynucleotide kinase/phosphatase (PNKP), a DNA repair enzyme that plays a critical role in rejoining DNA single- and double-strand breaks. For this purpose, newly identified hit compounds with potent PNKP inhibitory activity, imidopiperidines A12B4C50 and A83B4C63 were encapsulated in polymeric micelles of different poly(ethylene oxide)-b-poly(e-caprolactone) (PEO-b-PCL)-based structures. Our results showed efficient loading of A12B4C50 and A83B4C63 in PEO-b-PCLs with pendent carboxyl and benzyl carboxylate groups, respectively, and relatively slow release over 24 h. Both fre...
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Nanoencapsulation of Novel Inhibitors of PNKP for Selective Sensitization to Ionizing Radiation and Irinotecan and Induction of Synthetic Lethality
2018Co-Authors: Zahra Shire, Timothy D R Morgan, Mohammad Reza Vakili, Dennis G. Hall, Afsaneh Lavasanifar, Michael WeinfeldAbstract:There is increasing interest in developing and applying DNA repair inhibitors in cancer treatment to augment the efficacy of radiation and conventional genotoxic chemotherapy. However, targeting the inhibitor is required to avoid reducing the repair capacity of normal tissue. The aim of this study was to develop nanodelivery systems for the encapsulation of novel imidopiperidine-based inhibitors of the DNA 3′-phosphatase activity of polynucleotide kinase/phosphatase (PNKP), a DNA repair enzyme that plays a critical role in rejoining DNA single- and double-strand breaks. For this purpose, newly identified hit compounds with potent PNKP inhibitory activity, imidopiperidines A12B4C50 and A83B4C63 were encapsulated in polymeric micelles of different poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL)-based structures. Our results showed efficient loading of A12B4C50 and A83B4C63 in PEO-b-PCLs with pendent carboxyl and benzyl carboxylate groups, respectively, and relatively slow release over 24 h. Both free and encapsulated inhibitors were able to sensitize HCT116 cells to radiation and the topoisomerase I poison, irinotecan. In addition, the encapsulated inhibitors were capable of inducing synthetic lethalilty in phosphatase and tensin homologue (PTEN)-deficient cells. We also established the validity of the peptide GE11 as a suitable ligand for active targeted delivery of nanoencapsulated drugs to colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR). Our results show the potential of nanoencapsulated inhibitors of PNKP as either mono or combined therapeutic agents for colorectal cancer
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the rev1 interacting region rir motif in the scaffold protein xrcc1 mediates a low affinity interaction with polynucleotide kinase phosphatase PNKP during dna single strand break repair
Journal of Biological Chemistry, 2017Co-Authors: Claire Breslin, Rajam S Mani, Mesfin Fanta, Michael Weinfeld, Nicolas C Hoch, Keith W CaldecottAbstract:The scaffold protein X-ray repair cross-complementing 1 (XRCC1)interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair(SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase(PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay(MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylationdependent interaction site in XRCC1 and a fork-head associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved REV1-interacting (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified highaffinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
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the rev1 interacting region rir motif in the scaffold protein xrcc1 mediates a low affinity interaction with polynucleotide kinase phosphatase PNKP during dna single strand break repair
Journal of Biological Chemistry, 2017Co-Authors: Claire Breslin, Rajam S Mani, Mesfin Fanta, Michael Weinfeld, Nicolas C Hoch, Keith W CaldecottAbstract:The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
Keith W Caldecott - One of the best experts on this subject based on the ideXlab platform.
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the rev1 interacting region rir motif in the scaffold protein xrcc1 mediates a low affinity interaction with polynucleotide kinase phosphatase PNKP during dna single strand break repair
Journal of Biological Chemistry, 2017Co-Authors: Claire Breslin, Rajam S Mani, Mesfin Fanta, Michael Weinfeld, Nicolas C Hoch, Keith W CaldecottAbstract:The scaffold protein X-ray repair cross-complementing 1 (XRCC1)interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair(SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase(PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay(MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylationdependent interaction site in XRCC1 and a fork-head associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved REV1-interacting (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified highaffinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
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the rev1 interacting region rir motif in the scaffold protein xrcc1 mediates a low affinity interaction with polynucleotide kinase phosphatase PNKP during dna single strand break repair
Journal of Biological Chemistry, 2017Co-Authors: Claire Breslin, Rajam S Mani, Mesfin Fanta, Michael Weinfeld, Nicolas C Hoch, Keith W CaldecottAbstract:The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
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versatility in phospho dependent molecular recognition of the xrcc1 and xrcc4 dna damage scaffolds by aprataxin family fha domains
DNA Repair, 2015Co-Authors: Amy L Cherry, Keith W Caldecott, Timothy J Nott, Geoffrey Kelly, Stuart L Rulten, Stephen J SmerdonAbstract:Aprataxin, aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.
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impact of PNKP mutations associated with microcephaly seizures and developmental delay on enzyme activity and dna strand break repair
Nucleic Acids Research, 2012Co-Authors: John J Reynolds, Alexandra K Walker, Edward C Gilmore, Christopher A Walsh, Keith W CaldecottAbstract:Microcephaly with early-onset, intractable seizures and developmental delay (MCSZ) is a hereditary disease caused by mutations in polynucleotide kinase/phosphatase (PNKP), a DNA strand break repair protein with DNA 5'-kinase and DNA 3'-phosphatase activity. To investigate the molecular basis of this disease, we examined the impact of MCSZ mutations on PNKP activity in vitro and in cells. Three of the four mutations currently associated with MCSZ greatly reduce or ablate DNA kinase activity of recombinant PNKP at 30°C (L176F, T424Gfs48X and exon15Δfs4X), but only one of these mutations reduces DNA phosphatase activity under the same conditions (L176F). The fourth mutation (E326K) has little impact on either DNA kinase or DNA phosphatase activity at 30°C, but is less stable than the wild-type enzyme at physiological temperature. Critically, all of the MCSZ mutations identified to date result in ∼10-fold reduced cellular levels of PNKP protein, and reduced rates of chromosomal DNA strand break repair. Together, these data suggest that all four known MCSZ mutations reduce the cellular stability and level of PNKP protein, with three mutations likely ablating cellular DNA 5'-kinase activity and all of the mutations greatly reducing cellular DNA 3'-phosphatase activity.
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on enzyme activity
2012Co-Authors: John J Reynolds, Edward C Gilmore, Christopher A Walsh, Ra K. Walker, Keith W CaldecottAbstract:Impact of PNKP mutations associated with microcephaly, seizures and developmental dela
Rajam S Mani - One of the best experts on this subject based on the ideXlab platform.
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a synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3 phosphatase PNKP for targeted therapy of pten deficient colorectal cancer
Journal of Controlled Release, 2021Co-Authors: Sams M A Sadat, Rajam S Mani, Feridoun Karimibusheri, Gary R Martin, Igor M Paiva, Zahra Shire, Forughalsadat Sanaee, Timothy D R Morgan, Marco Paladino, Frank R JirikAbstract:Abstract Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3′-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN−/− xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.
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domain analysis of PNKP xrcc1 interactions influence of genetic variants of xrcc1
Journal of Biological Chemistry, 2019Co-Authors: Rajam S Mani, Ismail Abdou, Mesfin Fanta, Inbal Mermershtain, Michael J. Hendzel, J Mark N Glover, Michael WeinfeldAbstract:Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.
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the rev1 interacting region rir motif in the scaffold protein xrcc1 mediates a low affinity interaction with polynucleotide kinase phosphatase PNKP during dna single strand break repair
Journal of Biological Chemistry, 2017Co-Authors: Claire Breslin, Rajam S Mani, Mesfin Fanta, Michael Weinfeld, Nicolas C Hoch, Keith W CaldecottAbstract:The scaffold protein X-ray repair cross-complementing 1 (XRCC1)interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair(SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase(PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay(MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylationdependent interaction site in XRCC1 and a fork-head associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved REV1-interacting (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified highaffinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
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the rev1 interacting region rir motif in the scaffold protein xrcc1 mediates a low affinity interaction with polynucleotide kinase phosphatase PNKP during dna single strand break repair
Journal of Biological Chemistry, 2017Co-Authors: Claire Breslin, Rajam S Mani, Mesfin Fanta, Michael Weinfeld, Nicolas C Hoch, Keith W CaldecottAbstract:The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.
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structural and functional characterization of the PNKP xrcc4 ligiv dna repair complex
Nucleic Acids Research, 2017Co-Authors: Daniel R Aceytuno, Rajam S Mani, Shujuan Fang, Michael Weinfeld, Cortt G Piett, Zahra Havalishahriari, Ross A Edwards, Martial Rey, Fatima Javed, Michal HammelAbstract:Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.
Raven H Huang - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of bacterial protein hen1 activating the ligase activity of bacterial protein PNKP for rna repair
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Pei Wang, Chio Mui Chan, David Christensen, Can Zhang, Kiruthika Selvadurai, Raven H HuangAbstract:Ribotoxins cleave essential RNAs for cell killing in vivo, and the bacterial polynucleotide kinase-phosphatase (PNKP)/hua enhancer 1 (Hen1) complex has been shown to repair ribotoxin-cleaved RNAs in vitro. Bacterial PNKP/Hen1 is distinguished from other RNA repair systems by performing 3′-terminal 2′-O-methylation during RNA repair, which prevents the repaired RNA from repeated cleavage at the same site. To ensure the opportunity of 2′-O-methylation by bacterial Hen1 during RNA repair and, therefore, maintain the quality of the repaired RNA, PNKP/Hen1 has evolved to require the participation of Hen1 in RNA ligation, because PNKP alone is unable to carry out the reaction despite possessing all signature motifs of an RNA ligase. However, the precise role of Hen1 in RNA ligation is unknown. Here, we present the crystal structure of an active RNA ligase consisting of the C-terminal half of PNKP (PNKP-C) and the N-terminal half of Hen1 (Hen1-N) from Clostridium thermocellum. The structure reveals that the N-terminal domain of Clostridium thermocellum (Cth) Hen1, shaped like a left hand, grabs the flexible insertion module of CthPNKP and locks its conformation via further interaction with the C-terminal addition module of CthPNKP. Formation of the CthPNKP-C/Hen1-N heterodimer creates a ligation pocket with a width for two strands of RNA, depth for two nucleotides, and the adenosine monophosphate (AMP)-binding pocket at the bottom. The structure, combined with functional analyses, provides insight into the mechanism of how Hen1 activates the RNA ligase activity of PNKP for RNA repair.
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probing the substrate specificity of the bacterial PNKP hen1 rna repair system using synthetic rnas
RNA, 2012Co-Authors: Can Zhang, Pei Wang, Chio Mui Chan, Raven H HuangAbstract:Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named PNKP and Hen1, form a stable complex and are able to repair ribotoxincleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial PNKP/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant PNKP/ Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial PNKP/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the PNKP/Hen1 repair system is proposed.