Non-Homologous end Joining Factors XLF, PAXX and DNA-PKcs Maintain the Neural Stem and Progenitor Cell Population

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XLF, DNA-PKcs and PAXX maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

Non-homologous end joining factors XLF, PAXX and DNA-PKcs support neural stem and progenitor cells development

10.1101/2020.12.01.406629 ◽

2020 ◽

Author(s):

Raquel Gago-Fuentes ◽

Valentyn Oksenych

Keyword(s):

Mammalian Cells ◽

Neuronal Apoptosis ◽

Embryonic Lethality ◽

End Joining ◽

Double Knockout ◽

Dna Damages ◽

The Central Nervous System ◽

AbstractNon-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking core NHEJ factors Ku70 and Ku80. Inactivation of other core NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of one accessory NHEJ gene,e.g. Paxx, Mri and Dna-pkcs, results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of accessory NHEJ on early stages of neurodevelopment, we isolated neural stem and progenitors cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf,Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that accessory NHEJ factors are important for maintaining the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

Non-Homologous End Joining Factors XLF, PAXX and DNA-PKcs Maintain the Neural Stem and Progenitor Cell Population

Biomolecules ◽

10.3390/biom11010020 ◽

2020 ◽

Vol 11 (1) ◽

pp. 20

Author(s):

Raquel Gago-Fuentes ◽

Valentyn Oksenych

Keyword(s):

Mammalian Cells ◽

Progenitor Cell ◽

Neuronal Apoptosis ◽

Embryonic Lethality ◽

End Joining ◽

Double Knockout ◽

Dependent Protein ◽

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damage throughout the cell cycle and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking NHEJ factors Ku70 and Ku80. Inactivation of other NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of either Paxx, Mri or Dna-pkcs NHEJ gene results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of NHEJ factors on the early stages of neurodevelopment, we isolated neural stem and progenitor cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that XRCC4-like factor (XLF), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX) maintain the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

Non-Homologous end Joining Factors XLF, PAXX and DNA-PKcs are Required to Maintain the Neural Stem and Progenitor Cell Population

10.20944/preprints202012.0003.v1 ◽

2020 ◽

Author(s):

Raquel Gago-Fuentes ◽

Valentyn Oksenych

Keyword(s):

Mammalian Cells ◽

Progenitor Cell ◽

Neuronal Apoptosis ◽

Embryonic Lethality ◽

End Joining ◽

Double Knockout ◽

Dna Damages ◽

The Central Nervous System ◽

Non-homologous end-joining (NHEJ) is a major DNA repair pathway in mammalian cells that recognizes, processes and fixes DNA damages throughout the cell cycle, and is specifically important for homeostasis of post-mitotic neurons and developing lymphocytes. Neuronal apoptosis increases in the mice lacking core NHEJ factors Ku70 and Ku80. Inactivation of other core NHEJ genes, either Xrcc4 or Lig4, leads to massive neuronal apoptosis in the central nervous system (CNS) that correlates with embryonic lethality in mice. Inactivation of one accessory NHEJ gene, e.g. Paxx, Mri and Dna-pkcs, results in normal CNS development due to compensatory effects of Xlf. Combined inactivation of Xlf/Paxx, Xlf/Mri and Xlf/Dna-pkcs, however, results in late embryonic lethality and high levels of apoptosis in CNS. To determine the impact of accessory NHEJ on early stages of neurodevelopment, we isolated neural stem and progenitors cells from mouse embryos and investigated proliferation, self-renewal and differentiation capacity of these cells lacking either Xlf, Paxx, Dna-pkcs, Xlf/Paxx or Xlf/Dna-pkcs. We found that accessory NHEJ factors are important for maintaining the neural stem and progenitor cell populations and neurodevelopment in mammals, which is particularly evident in the double knockout models.

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Nature Communications ◽

10.1038/s41467-021-24486-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

Lalith Perera ◽

David D. Shock ◽

William A. Beard ◽

...

Keyword(s):

Mammalian Cells ◽

Time Lapse ◽

Strand Break ◽

Structural Basis ◽

End Joining ◽

Strand Breaks ◽

Nucleotide Pools ◽

Nucleotide Insertion ◽

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance

Blood ◽

10.1182/blood-2010-04-281071 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3197-3207 ◽

Cited By ~ 80

Author(s):

Kirsteen J. Campbell ◽

Mary L. Bath ◽

Marian L. Turner ◽

Cassandra J. Vandenberg ◽

Philippe Bouillet ◽

...

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Cytotoxic Agents ◽

Hematopoietic Stem ◽

Fetal Liver Cells ◽

Follicular Lymphomas ◽

Abstract Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eμ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eμ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

Anti-silencing factor 1A is associated with genome stability maintenance of mouse preimplantation embryos†

Biology of Reproduction ◽

10.1093/biolre/ioaa001 ◽

2020 ◽

Vol 102 (4) ◽

pp. 817-827

Author(s):

Kai Deng ◽

Wanyou Feng ◽

Xiaohua Liu ◽

Xiaoping Su ◽

Erwei Zuo ◽

...

Keyword(s):

Embryonic Development ◽

Genome Stability ◽

Mouse Embryos ◽

Preimplantation Embryos ◽

End Joining ◽

Dna Damages ◽

Developmental Potential ◽

Damage Repair ◽

Early Mouse

Abstract Genome stability is critical for the normal development of preimplantation embryos, as DNA damages may result in mutation and even embryo lethality. Anti-silencing factor 1A (ASF1A) is a histone chaperone and enriched in the MII oocytes as a maternal factor, which may be associated with the maintenance of genome stability. Thus, this study was undertaken to explore the role of ASF1A in maintaining the genome stability of early mouse embryos. The ASF1A expressed in the preimplantation embryos and displayed a dynamic pattern throughout the early embryonic development. Inhibition of ASF1A expression decreased embryonic development and increased DNA damages. Overexpression of ASF1A improved the developmental potential and decreased DNA damages. When 293T cells that had been integrated with RGS-NHEJ were co-transfected with plasmids of pcDNA3.1-ASF1A, gRNA-NHEJ, and hCas9, less cells expressed eGFP, indicating that non-homologous end joining was reduced by ASF1A. When 293T cells were co-transfected with plasmids of HR-donor, gRNA-HR, hCas9, and pcDNA3.1-ASF1A, more cells expressed eGFP, indicating that homologous recombination (HR) was enhanced by ASF1A. These results indicate that ASF1A may be associated with the genome stability maintenance of early mouse embryos and this action may be mediated by promoting DNA damage repair through HR pathway.

DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining

Nucleic Acids Research ◽

10.1093/nar/gkn450 ◽

2008 ◽

Vol 36 (15) ◽

pp. 4872-4882 ◽

Cited By ~ 56

Author(s):

S. Malyarchuk ◽

R. Castore ◽

L. Harrison

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

End Joining ◽

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m110.123844 ◽

2010 ◽

Vol 285 (30) ◽

pp. 23198-23207 ◽

Cited By ~ 13

Author(s):

Jia Liu ◽

Alokes Majumdar ◽

Jilan Liu ◽

Lawrence H. Thompson ◽

Michael M. Seidman

Keyword(s):

Mammalian Cells ◽

Single Strand ◽

End Joining ◽

DNA double-strand break repair within heterochromatic regions

Biochemical Society Transactions ◽

10.1042/bst20110631 ◽

2012 ◽

Vol 40 (1) ◽

pp. 173-178 ◽

Cited By ~ 27

Author(s):

Johanne M. Murray ◽

Tom Stiff ◽

Penny A. Jeggo

Keyword(s):

Chromatin Structure ◽

Mammalian Cells ◽

Strand Break ◽

Higher Order ◽

End Joining ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Dna Dsbs ◽

A Cell ◽

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.