The Experts below are selected from a list of 1293 Experts worldwide ranked by ideXlab platform
Vilhelm A Bohr - One of the best experts on this subject based on the ideXlab platform.
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interaction between RECQL4 and ogg1 promotes repair of oxidative base lesion 8 oxog and is regulated by sirt1 deacetylase
Nucleic Acids Research, 2020Co-Authors: Shunlei Duan, Deborah L Croteau, Vilhelm A Bohr, Xuerui Han, Mansour Akbari, Lene Juel RasmussenAbstract:OGG1 initiated base excision repair (BER) is the major pathway for repair of oxidative DNA base damage 8-oxoguanine (8-oxoG). Here, we report that RECQL4 DNA helicase, deficient in the cancer-prone and premature aging Rothmund-Thomson syndrome, physically and functionally interacts with OGG1. RECQL4 promotes catalytic activity of OGG1 and RECQL4 deficiency results in defective 8-oxoG repair and increased genomic 8-oxoG. Furthermore, we show that acute oxidative stress leads to increased RECQL4 acetylation and its interaction with OGG1. The NAD+-dependent protein SIRT1 deacetylates RECQL4 in vitro and in cells thereby controlling the interaction between OGG1 and RECQL4 after DNA repair and maintaining RECQL4 in a low acetylated state. Collectively, we find that RECQL4 is involved in 8-oxoG repair through interaction with OGG1, and that SIRT1 indirectly modulates BER of 8-oxoG by controlling RECQL4-OGG1 interaction.
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cell cycle dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in dna double strand break repair
Nature Communications, 2017Co-Authors: Raghavendra A Shamanna, Prabhat Khadka, Deborah L Croteau, Tomasz Kulikowicz, Jessica K De Freitas, Mustafa Nazir Okur, Priscella P Holland, Jane Tian, Anthony J Davis, Vilhelm A BohrAbstract:Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.
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recql5 has unique strand annealing properties relative to the other human recq helicase proteins
DNA Repair, 2016Co-Authors: Prabhat Khadka, Deborah L Croteau, Vilhelm A BohrAbstract:The RecQ helicases play important roles in genome maintenance and DNA metabolism (replication, recombination, repair, and transcription). Five different homologs are present in humans, three of which are implicated in accelerated aging genetic disorders: Rothmund Thomson (RECQL4), Werner (WRN), and Bloom (BLM) syndromes. While the DNA helicase activities of the 5 human RecQ helicases have been extensively characterized, much less is known about their DNA double strand annealing activities. Strand annealing is an important integral enzymatic activity in DNA metabolism, including DNA repair. Here, we have characterized the strand annealing activities of all five human RecQ helicase proteins and compared them. Interestingly, the relative strand annealing activities of the five RecQ proteins are not directly (inversely) related to their helicase activities. RECQL5 possesses relatively strong annealing activity on long or small duplexed substrates compared to the other RecQs. Additionally, the strand annealing activity of RECQL5 is not inhibited by the presence of ATP, unlike the other RecQs. We also show that RECQL5 efficiently catalyzes annealing of RNA to DNA in vitro in the presence or absence of ATP, revealing a possible new function for RECQL5. Additionally, we investigate how different known RecQ interacting proteins, RPA, Ku, FEN1 and RAD51, regulate their strand annealing activity. Collectively, we find that the human RecQ proteins possess differential DNA double strand annealing activities and we speculate on their individual roles in DNA repair. This insight is important in view of the many cellular DNA metabolic actions of the RecQ proteins and elucidates their unique functions in the cell.
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recq helicase RECQL4 participates in non homologous end joining and interacts with the ku complex
Carcinogenesis, 2014Co-Authors: Raghavendra A Shamanna, Dharmendra Kumar Singh, Huiming Lu, Gladys Mirey, Guido Keijzers, Bernard Salles, Deborah L Croteau, Vilhelm A BohrAbstract:RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.
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senescence induced by RECQL4 dysfunction contributes to rothmund thomson syndrome features in mice
Cell Death and Disease, 2014Co-Authors: Evandro Fei Fang, Deborah L Croteau, Peter Sykora, Tomasz Kulikowicz, Yongqing Zhang, Kevin G Becker, Vilhelm A BohrAbstract:Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16INK4a or/and p21WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, RECQL4-deficient mice (RECQL4HD). Tail fibroblasts from RECQL4HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in RECQL4HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.
Carl R Walkley - One of the best experts on this subject based on the ideXlab platform.
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rothmund thomson syndrome like RECQL4 truncating mutations cause a haploinsufficient low bone mass phenotype in mice
Molecular and Cellular Biology, 2020Co-Authors: Monique Smeets, Carl R Walkley, Natalie A Sims, Wilson Castillotandazo, Ann E FrazierAbstract:Rothmund-Thomson Syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germline compound bi-allelic protein-truncating mutations of RECQL4 As existing murine models employ RECQL4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating RECQL4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4, and resulted in homozygous embryonic lethality and a haploinsufficient low bone mass phenotype. Combination of a truncating mutation with a conditional RECQL4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine RECQL4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.
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rothmund thomson syndrome like RECQL4 truncating mutations cause a haploinsufficient low bone mass phenotype in mice
bioRxiv, 2020Co-Authors: Monique Smeets, Carl R Walkley, Natalie A Sims, Wilson Castillotandazo, Ann E FrazierAbstract:Rothmund-Thomson Syndrome (RTS) is an autosomal recessive disorder characterized by poikiloderma, sparse or absent hair, and defects in the skeletal system such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients typically present with germline compound bi-allelic protein-truncating mutations of RECQL4. As existing murine models predominantly employ RECQL4 null alleles, we have here attempted to more accurately model the mutational spectrum of RTS by generating mice with patient-mimicking truncating RECQL4 mutations. We found that truncating mutations impaired stability and subcellular localization of RECQL4, which translated to a homozygous embryonic lethality and haploinsufficient low bone mass and reduced cortical bone thickness phenotypes. Combination of a truncating mutation with a conditional RECQL4 null allele demonstrated that these defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis, even after exposure to irradiation. We also utilized murine RECQL4 null cells to assess the impact of a wider range of human RECQL4 mutations using an in vitro complementation assay. We found differential effects of distinct RECQL4 mutations. While some created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations lead to the development of an osteoporosis-like phenotype through defects in early osteoblast progenitors in mice and identify RECQL4 gene dosage as a novel regulator of bone mass.
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3059 protein truncating but not helicase inactivating RECQL4 mutations cause bone marrow failure
Experimental Hematology, 2020Co-Authors: Wilson Castillotandazo, Monique Smeets, Carl R WalkleyAbstract:Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash, skeletal dysplasias, premature ageing, and caused by mutations in RECQL4. RTS is a familial cancer syndrome, with hematological cancers amongst the most common reported malignancies. We previously reported that mice with somatic deletion of RECQL4, resulting in null alleles, developed a fully penetrant bone marrow failure syndrome. RTS patients, however, have truncating mutations that result in hypomorphic alleles, not null alleles. To understand the impact of RECQL4 mutations on haematopoiesis, we established mice with three distinct point mutations: K525A, which results in an ATP-dependent helicase-inactive but full-length protein, and G522EfsX43 and R347X, which result in truncated protein products. We crossed the point mutant mice to the R26-CreER RECQL4 fl/fl line, and at 8-10 weeks of age fed tamoxifen containing food for 30 days to delete the wild-type RECQL4 floxed allele leaving only the mutation expressed. Analysis of peripheral blood and bone marrow by FACS showed that only mice carrying truncating mutations developed bone marrow failure. To validate these results in vitro, we immortalized R26-CreER RECQL4 point mutant cells with Hoxb8 retrovirus and treated them with tamoxifen for 14 days. In this setting, we also found that truncating mutations caused a marked decrease in cell proliferation, while the helicase-inactive mutation did not. Finally, we performed in vitro complementation assays to determine the capacity of human RECQL4 mutations, similar to our murine alleles, to rescue Hoxb8 R26-CreER RECQL4-depleted myeloid cells. Results showed that the helicase-inactive mutant successfully rescued the RECQL4-depleted cells, while the truncating human mutants did not. Collectively, these data demonstrate that the helicase function of RECQL4 is dispensable for hematopoiesis but that the deletion of both the helicase and the C-terminal domains in truncating mutations has deleterious effects on hematopoietic cells.
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RESEARCH ARTICLE The DNA Helicase RECQL4 Is Required for Normal Osteoblast Expansion and Osteosarcoma Formation
2016Co-Authors: Mannu K Walia, Louise E Purton, Monique F Smeets, Anthony J Mutsaers, Nicole C Walsh, John T Martin, A. Sims, Carl R WalkleyAbstract:RECQL4mutations are associated with Rothmund Thomson Syndrome (RTS), RAPADI-LINO Syndrome and Baller-Gerold Syndrome. These patients display a range of benign skeletal abnormalities such as low bone mass. In addition, RTS patients have a highly in-creased incidence of osteosarcoma (OS). The role of RECQL4 in normal adult bone devel-opment and homeostasis is largely uncharacterized and how mutation of RECQL4 contributes to OS susceptibility is not known. We hypothesised that RECQL4 was required for normal skeletal development and both benign and malignant osteoblast function, which we have tested in the mouse. RECQL4 deletion in vivo at the osteoblastic progenitor stage of dif-ferentiation resulted in mice with shorter bones and reduced bone volume, assessed at 9 weeks of age. This was associated with an osteoblast intrinsic decrease in mineral apposi-tion rate and bone formation rate in the RECQL4-deficient cohorts. Deletion of RECQL4 in ma-ture osteoblasts/osteocytes in vivo, however, did not cause a detectable phenotype. Acute deletion of RECQL4 in primary osteoblasts or shRNA knockdown in an osteoblastic cell line caused failed proliferation, accompanied by cell cycle arrest, induction of apoptosis and im
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The DNA helicase RECQL4 is required for normal osteoblast expansion and osteosarcoma formation.
Public Library of Science (PLoS), 2015Co-Authors: Mannu K Walia, Louise E Purton, Monique F Smeets, Anthony J Mutsaers, Natalie A Sims, Nicole C Walsh, John T Martin, Carl R WalkleyAbstract:RECQL4 mutations are associated with Rothmund Thomson Syndrome (RTS), RAPADILINO Syndrome and Baller-Gerold Syndrome. These patients display a range of benign skeletal abnormalities such as low bone mass. In addition, RTS patients have a highly increased incidence of osteosarcoma (OS). The role of RECQL4 in normal adult bone development and homeostasis is largely uncharacterized and how mutation of RECQL4 contributes to OS susceptibility is not known. We hypothesised that RECQL4 was required for normal skeletal development and both benign and malignant osteoblast function, which we have tested in the mouse. RECQL4 deletion in vivo at the osteoblastic progenitor stage of differentiation resulted in mice with shorter bones and reduced bone volume, assessed at 9 weeks of age. This was associated with an osteoblast intrinsic decrease in mineral apposition rate and bone formation rate in the RECQL4-deficient cohorts. Deletion of RECQL4 in mature osteoblasts/osteocytes in vivo, however, did not cause a detectable phenotype. Acute deletion of RECQL4 in primary osteoblasts or shRNA knockdown in an osteoblastic cell line caused failed proliferation, accompanied by cell cycle arrest, induction of apoptosis and impaired differentiation. When cohorts of animals were aged long term, the loss of RECQL4 alone was not sufficient to initiate OS. We then crossed the RECQL4fl/fl allele to a fully penetrant OS model (Osx-Cre p53fl/fl). Unexpectedly, the Osx-Cre p53fl/flRECQL4fl/fl (dKO) animals had a significantly increased OS-free survival compared to Osx-Cre p53fl/fl or Osx-Cre p53fl/flRECQL4fl/+ (het) animals. The extended survival was explained when the RECQL4 status in the tumors that arose was assessed, and in no case was there complete deletion of RECQL4 in the dKO OS. These data provide a mechanism for the benign skeletal phenotypes of RECQL4 mutation syndromes. We propose that tumor suppression and osteosarcoma susceptibility are most likely a function of mutant, not null, alleles of RECQL4
Deborah L Croteau - One of the best experts on this subject based on the ideXlab platform.
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interaction between RECQL4 and ogg1 promotes repair of oxidative base lesion 8 oxog and is regulated by sirt1 deacetylase
Nucleic Acids Research, 2020Co-Authors: Shunlei Duan, Deborah L Croteau, Vilhelm A Bohr, Xuerui Han, Mansour Akbari, Lene Juel RasmussenAbstract:OGG1 initiated base excision repair (BER) is the major pathway for repair of oxidative DNA base damage 8-oxoguanine (8-oxoG). Here, we report that RECQL4 DNA helicase, deficient in the cancer-prone and premature aging Rothmund-Thomson syndrome, physically and functionally interacts with OGG1. RECQL4 promotes catalytic activity of OGG1 and RECQL4 deficiency results in defective 8-oxoG repair and increased genomic 8-oxoG. Furthermore, we show that acute oxidative stress leads to increased RECQL4 acetylation and its interaction with OGG1. The NAD+-dependent protein SIRT1 deacetylates RECQL4 in vitro and in cells thereby controlling the interaction between OGG1 and RECQL4 after DNA repair and maintaining RECQL4 in a low acetylated state. Collectively, we find that RECQL4 is involved in 8-oxoG repair through interaction with OGG1, and that SIRT1 indirectly modulates BER of 8-oxoG by controlling RECQL4-OGG1 interaction.
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cell cycle dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in dna double strand break repair
Nature Communications, 2017Co-Authors: Raghavendra A Shamanna, Prabhat Khadka, Deborah L Croteau, Tomasz Kulikowicz, Jessica K De Freitas, Mustafa Nazir Okur, Priscella P Holland, Jane Tian, Anthony J Davis, Vilhelm A BohrAbstract:Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.
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recql5 has unique strand annealing properties relative to the other human recq helicase proteins
DNA Repair, 2016Co-Authors: Prabhat Khadka, Deborah L Croteau, Vilhelm A BohrAbstract:The RecQ helicases play important roles in genome maintenance and DNA metabolism (replication, recombination, repair, and transcription). Five different homologs are present in humans, three of which are implicated in accelerated aging genetic disorders: Rothmund Thomson (RECQL4), Werner (WRN), and Bloom (BLM) syndromes. While the DNA helicase activities of the 5 human RecQ helicases have been extensively characterized, much less is known about their DNA double strand annealing activities. Strand annealing is an important integral enzymatic activity in DNA metabolism, including DNA repair. Here, we have characterized the strand annealing activities of all five human RecQ helicase proteins and compared them. Interestingly, the relative strand annealing activities of the five RecQ proteins are not directly (inversely) related to their helicase activities. RECQL5 possesses relatively strong annealing activity on long or small duplexed substrates compared to the other RecQs. Additionally, the strand annealing activity of RECQL5 is not inhibited by the presence of ATP, unlike the other RecQs. We also show that RECQL5 efficiently catalyzes annealing of RNA to DNA in vitro in the presence or absence of ATP, revealing a possible new function for RECQL5. Additionally, we investigate how different known RecQ interacting proteins, RPA, Ku, FEN1 and RAD51, regulate their strand annealing activity. Collectively, we find that the human RecQ proteins possess differential DNA double strand annealing activities and we speculate on their individual roles in DNA repair. This insight is important in view of the many cellular DNA metabolic actions of the RecQ proteins and elucidates their unique functions in the cell.
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recq helicase RECQL4 participates in non homologous end joining and interacts with the ku complex
Carcinogenesis, 2014Co-Authors: Raghavendra A Shamanna, Dharmendra Kumar Singh, Huiming Lu, Gladys Mirey, Guido Keijzers, Bernard Salles, Deborah L Croteau, Vilhelm A BohrAbstract:RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.
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senescence induced by RECQL4 dysfunction contributes to rothmund thomson syndrome features in mice
Cell Death and Disease, 2014Co-Authors: Evandro Fei Fang, Deborah L Croteau, Peter Sykora, Tomasz Kulikowicz, Yongqing Zhang, Kevin G Becker, Vilhelm A BohrAbstract:Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16INK4a or/and p21WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, RECQL4-deficient mice (RECQL4HD). Tail fibroblasts from RECQL4HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in RECQL4HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.
Dharmendra Kumar Singh - One of the best experts on this subject based on the ideXlab platform.
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recq helicase RECQL4 participates in non homologous end joining and interacts with the ku complex
Carcinogenesis, 2014Co-Authors: Raghavendra A Shamanna, Dharmendra Kumar Singh, Huiming Lu, Gladys Mirey, Guido Keijzers, Bernard Salles, Deborah L Croteau, Vilhelm A BohrAbstract:RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.
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RECQL4 in genomic instability and aging
Trends in Genetics, 2012Co-Authors: Deborah L Croteau, Leslie K. Ferrarelli, Dharmendra Kumar Singh, Vilhelm A BohrAbstract:Helicases are ubiquitous proteins that unwind DNA and participate in DNA metabolism including replication, repair, transcription, and chromatin organization. The highly conserved RecQ helicase family proteins are important in these transactions and have been termed the guardians of the genome. Humans have five members of this family: WRN, BLM, RECQL4, RECQL1, and RECQL5. The first three of are associated with premature aging and cancer prone syndromes, but the latter two proteins have not yet been implicated in any human disease. Although WRN and BLM have been fairly well characterized, RECQL4 has only recently been intensively investigated. The sum of this work to date has shown that RECQL4 has helicase activity and localizes to telomeres and mitochondria. In addition, new protein partners are emerging, implicating RECQL4 in novel processes. Here, we describe these recent findings which place RECQL4 at the crossroads of genomic instability and aging processes.
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RECQL4 localizes to mitochondria and preserves mitochondrial dna integrity
Aging Cell, 2012Co-Authors: Deborah L Croteau, Chandrika Canugovi, Dharmendra Kumar Singh, Zhengming Wang, Marie L Rossi, Mahesh Ramamoorthy, Peter Sykora, Mansour Akbari, Jane Tian, Rajesh KasiviswanathanAbstract:RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability, and cancer predisposition. RECQL4 is a member of the RecQ helicase family, and has many similarities to WRN protein, which is also implicated in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A, and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4's helicase activity. RECQL4 is the first 3'-5' RecQ helicase to be found in both human and mouse mitochondria, and the loss of RECQL4 alters mitochondrial integrity.
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The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability
Nucleic Acids Research, 2012Co-Authors: Dharmendra Kumar Singh, Venkateswarlu Popuri, Avik K. Ghosh, Deborah L Croteau, Pavel Janscak, Marie L Rossi, Mahesh Ramamoorthy, Tomasz Kulikowicz, Igor Shevelev, Vilhelm A BohrAbstract:Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.
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RECQL4 the protein mutated in rothmund thomson syndrome functions in telomere maintenance
Journal of Biological Chemistry, 2012Co-Authors: Avik K. Ghosh, Dharmendra Kumar Singh, Deborah L Croteau, Marie L Rossi, Christopher A Dunn, Mahesh Ramamoorthy, Yie Liu, Vilhelm A BohrAbstract:Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.
Wilson Castillotandazo - One of the best experts on this subject based on the ideXlab platform.
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rothmund thomson syndrome like RECQL4 truncating mutations cause a haploinsufficient low bone mass phenotype in mice
Molecular and Cellular Biology, 2020Co-Authors: Monique Smeets, Carl R Walkley, Natalie A Sims, Wilson Castillotandazo, Ann E FrazierAbstract:Rothmund-Thomson Syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germline compound bi-allelic protein-truncating mutations of RECQL4 As existing murine models employ RECQL4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating RECQL4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4, and resulted in homozygous embryonic lethality and a haploinsufficient low bone mass phenotype. Combination of a truncating mutation with a conditional RECQL4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine RECQL4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.
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rothmund thomson syndrome like RECQL4 truncating mutations cause a haploinsufficient low bone mass phenotype in mice
bioRxiv, 2020Co-Authors: Monique Smeets, Carl R Walkley, Natalie A Sims, Wilson Castillotandazo, Ann E FrazierAbstract:Rothmund-Thomson Syndrome (RTS) is an autosomal recessive disorder characterized by poikiloderma, sparse or absent hair, and defects in the skeletal system such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients typically present with germline compound bi-allelic protein-truncating mutations of RECQL4. As existing murine models predominantly employ RECQL4 null alleles, we have here attempted to more accurately model the mutational spectrum of RTS by generating mice with patient-mimicking truncating RECQL4 mutations. We found that truncating mutations impaired stability and subcellular localization of RECQL4, which translated to a homozygous embryonic lethality and haploinsufficient low bone mass and reduced cortical bone thickness phenotypes. Combination of a truncating mutation with a conditional RECQL4 null allele demonstrated that these defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis, even after exposure to irradiation. We also utilized murine RECQL4 null cells to assess the impact of a wider range of human RECQL4 mutations using an in vitro complementation assay. We found differential effects of distinct RECQL4 mutations. While some created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations lead to the development of an osteoporosis-like phenotype through defects in early osteoblast progenitors in mice and identify RECQL4 gene dosage as a novel regulator of bone mass.
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3059 protein truncating but not helicase inactivating RECQL4 mutations cause bone marrow failure
Experimental Hematology, 2020Co-Authors: Wilson Castillotandazo, Monique Smeets, Carl R WalkleyAbstract:Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash, skeletal dysplasias, premature ageing, and caused by mutations in RECQL4. RTS is a familial cancer syndrome, with hematological cancers amongst the most common reported malignancies. We previously reported that mice with somatic deletion of RECQL4, resulting in null alleles, developed a fully penetrant bone marrow failure syndrome. RTS patients, however, have truncating mutations that result in hypomorphic alleles, not null alleles. To understand the impact of RECQL4 mutations on haematopoiesis, we established mice with three distinct point mutations: K525A, which results in an ATP-dependent helicase-inactive but full-length protein, and G522EfsX43 and R347X, which result in truncated protein products. We crossed the point mutant mice to the R26-CreER RECQL4 fl/fl line, and at 8-10 weeks of age fed tamoxifen containing food for 30 days to delete the wild-type RECQL4 floxed allele leaving only the mutation expressed. Analysis of peripheral blood and bone marrow by FACS showed that only mice carrying truncating mutations developed bone marrow failure. To validate these results in vitro, we immortalized R26-CreER RECQL4 point mutant cells with Hoxb8 retrovirus and treated them with tamoxifen for 14 days. In this setting, we also found that truncating mutations caused a marked decrease in cell proliferation, while the helicase-inactive mutation did not. Finally, we performed in vitro complementation assays to determine the capacity of human RECQL4 mutations, similar to our murine alleles, to rescue Hoxb8 R26-CreER RECQL4-depleted myeloid cells. Results showed that the helicase-inactive mutant successfully rescued the RECQL4-depleted cells, while the truncating human mutants did not. Collectively, these data demonstrate that the helicase function of RECQL4 is dispensable for hematopoiesis but that the deletion of both the helicase and the C-terminal domains in truncating mutations has deleterious effects on hematopoietic cells.
