PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

Published on Sep 20, 2016in Proceedings of the National Academy of Sciences of the United States of America9.58
· DOI :10.1073/pnas.1611882113
Estimated H-index: 1
(Harvard University),
Frederick W. Alt157
Estimated H-index: 157
(Harvard University),
Richard L. Frock14
Estimated H-index: 14
(Harvard University)
Abstract Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B–cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro–B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.
  • References (37)
  • Citations (34)
📖 Papers frequently viewed together
12 Authors (Takashi Ochi, ..., Stephen P. Jackson)
130 Citations
20167.82Cell Reports
13 Authors (Chloé Lescale, ..., Ludovic Deriano)
30 Citations
19 Authors (Mengtan Xing, ..., Dongyi Xu)
85 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
Abstract P53 is a transcription factor highly inducible by many stress signals such as DNA damage, oncogene activation, and nutrient deprivation. Cell-cycle arrest and apoptosis are the most prominent outcomes of p53 activation. Many studies showed that p53 cell-cycle and apoptosis functions are important for preventing tumor development. p53 also regulates many cellular processes including metabolism, antioxidant response, and DNA repair. Emerging evidence suggests that these noncanonical p53 a...
79 CitationsSource
#1Bjoern Schwer (Harvard University)H-Index: 23
#2Pei-Chi Wei (Harvard University)H-Index: 4
Last. Frederick W. Alt (Harvard University)H-Index: 157
view all 7 authors...
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global...
46 CitationsSource
#1Chloé Lescale (Pasteur Institute)H-Index: 5
#2Vincent Abramowski (Paris V: Paris Descartes University)H-Index: 2
Last. Ludovic Deriano (Pasteur Institute)H-Index: 18
view all 9 authors...
XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2 c/c mice), XLF deficiency leads to a profound lymphopenia associ...
26 CitationsSource
#1Pei-Chi Wei (Harvard University)H-Index: 4
#2Amelia N. Chang (Harvard University)H-Index: 3
Last. Bjoern Schwer (Harvard University)H-Index: 23
view all 7 authors...
Summary Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected onl...
103 CitationsSource
#1Alexandre OrthweinH-Index: 9
Last. Daniel DurocherH-Index: 56
view all 13 authors...
A mechanism for the repression of homologous recombination in G1, the stage of the cell cycle preceding replication, is determined; the critical aspects are the interaction between BRCA1 and PALB2–BRCA2, and suppression of DNA-end resection.
195 CitationsSource
#1Junchao DongH-Index: 6
Last. Frederick W. AltH-Index: 157
view all 15 authors...
High-throughput genome-wide sequencing reveals why class switch recombination in the IgH locus, an essential step in the process of antibody generation, has a directional joining bias towards deletion rather than inversion.
49 CitationsSource
#1Sunetra Roy (MSU: Michigan State University)H-Index: 3
Last. Katheryn Meek (MSU: Michigan State University)H-Index: 26
view all 8 authors...
The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA ends in vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNA in vitr...
29 CitationsSource
#1Nicholas C. Turner (The Royal Marsden NHS Foundation Trust)H-Index: 45
#2Jungsil RoH-Index: 42
Last. Massimo Cristofanilli (Thomas Jefferson University)H-Index: 70
view all 14 authors...
BackgroundGrowth of hormone-receptor–positive breast cancer is dependent on cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which promote progression from the G1 phase to the S phase of the cell cycle. We assessed the efficacy of palbociclib (an inhibitor of CDK4 and CDK6) and fulvestrant in advanced breast cancer. MethodsThis phase 3 study involved 521 patients with advanced hormone-receptor–positive, human epidermal growth factor receptor 2–negative breast cancer that had relapsed or progres...
551 CitationsSource
#1A CraxtonH-Index: 3
#2J SomersH-Index: 1
Last. Michal MalewiczH-Index: 5
view all 6 authors...
Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein a...
39 CitationsSource
#1Joyce K. Hwang (HHMI: Howard Hughes Medical Institute)H-Index: 4
#2Frederick W. Alt (HHMI: Howard Hughes Medical Institute)H-Index: 157
Last. Leng-Siew Yeap (HHMI: Howard Hughes Medical Institute)H-Index: 5
view all 3 authors...
The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies w...
51 CitationsSource
Cited By34
#1Xiaojing Liu (CAS: Chinese Academy of Sciences)
#2Tingting Liu (CAS: Chinese Academy of Sciences)H-Index: 1
Last. Liu Daisy Liu (CAS: Chinese Academy of Sciences)H-Index: 1
view all 20 authors...
Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturba...
#1Jennifer J Crowe (Columbia University)
#2Xiaobin Wang (Columbia University)
Last. Shan Zha (Columbia University)H-Index: 25
view all 6 authors...
The DNA dependent protein kinase (DNA-PK), composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. Naive B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends, and r...
#1Benjamin M. Stinson (Harvard University)H-Index: 1
#2Andrew T. Moreno (Harvard University)H-Index: 1
Last. Joseph J. Loparo (Harvard University)H-Index: 23
view all 4 authors...
Summary Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires t...
#1Sergio Castañeda-Zegarra (NTNU: Norwegian University of Science and Technology)H-Index: 1
#1Sergio Castaneda-Zegarra (NTNU: Norwegian University of Science and Technology)
Last. Valentyn Oksenych (NTNU: Norwegian University of Science and Technology)H-Index: 11
view all 4 authors...
Non-homologous end joining (NHEJ) is a DNA repair pathway required to detect, process and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is required for the V(D)J recombination in developing B and T lymphocytes. During the NHEJ, the core factors Ku70 and Ku80 form a heterodimer called Ku, which recognizes DSBs and promotes the recruitment of accessory factors (e.g., PAXX, Mri, Dna-pkcs, Artemis) and downstream core factors XLF, XRCC4, and Lig4. Mutations in ...
#1Demis Menolfi (Columbia University)H-Index: 5
#2Shan Zha (Columbia University)H-Index: 25
Genomic integrity is critical for normal development, healthy aging and suppressing oncogenic transformation. The DNA damage response (DDR) is a complex network that is activated by DNA structural changes to preserve genome integrity. Situated at the apex of the mammalian DDR are three PI3-kinase-related protein kinases—ATM, DNA-PKcs and ATR. They are activated by different DNA lesions via direct binding to their unique sensor protein complexes (MRE11-RAD50-NBS1 for ATM, Ku70-Ku80/86 for DNA-PKc...
#1S. Haihua ChuH-Index: 5
#2Jonathan ChabonH-Index: 1
Last. Putzer J. HungH-Index: 3
view all 17 authors...
Summary Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lys...
#1Yongfeng Yang (PKU: Peking University)H-Index: 2
#2Chuanzhen Yang (PKU: Peking University)H-Index: 2
Last. Xiaofeng Zheng (PKU: Peking University)H-Index: 19
view all 8 authors...
The DNA damage response (DDR) is essential for maintaining genome integrity. Mounting evidence reveals that protein modifications play vital roles in the DDR. Here, we show that USP38 is involved in the DDR by regulating the activity of HDAC1. In response to DNA damage, USP38 interacted with HDAC1 and specifically removed the K63-linked ubiquitin chain promoting the deacetylase activity of HDAC1. As a result, HDAC1 was able to deacetylate H3K56. USP38 deletion resulted in persistent focal accumu...
#1Mireille Bétermier (Université Paris-Saclay)H-Index: 23
#2Valérie Borde (University of Paris)H-Index: 2
Last. Jean-Pierre de Villartay (Paris V: Paris Descartes University)H-Index: 38
view all 3 authors...
DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the ‘price to pay’ to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome...
2 CitationsSource
#1Demis Menolfi (Columbia University)H-Index: 5
Last. Zha S
view all 2 authors...
DNA damage, especially DNA double strand breaks (DSBs) and replication stress, activates a complex post-translational network termed DNA damage response (DDR). Our review focuses on three PI3-kinase related protein kinases-ATM, ATR and DNA-PKcs, which situate at the apex of the mammalian DDR. They are recruited to and activated at the DNA damage sites by their respective sensor protein complexes-MRE11/RAD50/NBS1 for ATM, RPA/ATRIP for ATR and KU70-KU80/86 (XRCC6/XRCC5) for DNA-PKcs. Upon activat...
#1Stefania Musilli (French Institute of Health and Medical Research)
#2Vincent Abramowski (University of Paris)
Last. Jean-Pierre de Villartay (University of Paris)
view all 4 authors...
The repair of DNA double strand breaks (DSBs) by the non-homologous end-joining pathway (NHEJ) is central for the proper development of the adaptive immune system. This repair pathway involves eight factors, including XRCC4-like factor (XLF)/Cernunnos and the paralog of XRCC4 and XLF, PAXX non-homologous end-joining factor (PAXX). Xlf-/- and Paxx-/- mice are viable and exhibit only a mild immunophenotype. However, mice lacking both PAXX and XLF are embryonic lethal because postmitotic neurons un...