scholarly journals Deletion of the Nucleotide Excision Repair Gene Ercc1 Reduces Immunoglobulin Class Switching and Alters Mutations Near Switch Recombination Junctions

2004 ◽  
Vol 200 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Carol E. Schrader ◽  
Joycelyn Vardo ◽  
Erin Linehan ◽  
Michael Z. Twarog ◽  
Laura J. Niedernhofer ◽  
...  
Keyword(s):  
B Cells ◽  
Excision Repair ◽  
Dna Lesions ◽  
Wild Type ◽  
Repair Gene ◽  
Class Switch ◽  
Double Stranded Dna ◽  
Nucleotide Excision

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3′ single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1−/− splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Sμ segments and recombined Sμ-Sγ3 segments cloned from Ercc1−/− splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1−/− cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S–S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.

scholarly journals Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

2004 ◽  
Vol 200 (9) ◽  
pp. 1111-1121 ◽  
Author(s):  
Joanne M. Lumsden ◽  
Thomas McCarty ◽  
Lisa K. Petiniot ◽  
Rhuna Shen ◽  
Carrolee Barlow ◽  
...  
Keyword(s):  
B Cells ◽  
Dna Recombination ◽  
Class Switch Recombination ◽  
Intrinsic Defect ◽  
Wild Type ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Deficient Mice

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell–dependent antigen (Ag), Atm−/− mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm−/− B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm−/− cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm−/− B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm−/− B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm−/− B cells occurs at the level of genomic DNA recombination as measured by digestion–circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the μ–γ1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

scholarly journals Role for Mismatch Repair Proteins Msh2, Mlh1, and Pms2 in Immunoglobulin Class Switching Shown by Sequence Analysis of Recombination Junctions

2002 ◽  
Vol 195 (3) ◽  
pp. 367-373 ◽  
Author(s):  
Carol E. Schrader ◽  
Joycelyn Vardo ◽  
Janet Stavnezer
Keyword(s):  
B Cells ◽  
Mismatch Repair ◽  
Type B ◽  
Wild Type ◽  
Class Switching ◽  
Class Switch ◽  
Mmr Proteins

B cells from mice deficient in mismatch repair (MMR) proteins show decreased ability to undergo class switch recombination in vitro and in vivo. The deficit is not accompanied by any reduction in cell viability or alterations in the cell cycle in B cells cultured in vitro. To assess the role of MMR in switching we examined the nucleotide sequences of Sμ-Sγ3 recombination junctions in splenic B cells induced in culture to switch to IgG3. The data demonstrate clear differences in the sequences of switch junctions in wild-type B cells in comparison with Msh2-, Mlh1-, and Pms2-deficient B cells. Sequences of switch junctions from Msh2-deficient cells showed decreased lengths of microhomology between Sμ and Sγ3 relative to junctions from wild-type cells and an increase in insertions, i.e., nucleotides which do not appear to be derived from either the Sμ or Sγ3 parental sequence. By contrast, 23% of junctions from Mlh1- and Pms2-deficient cells occurred at unusually long stretches of microhomology. The data indicate that MMR proteins are directly involved in class switching and that the role of Msh2 differs from that of Mlh1 and Pms2.

scholarly journals Structural studies on M. tuberculosis O6-methylguanine methyltransferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C832-C832
Author(s):  
Menico Rizzi ◽  
Riccardo Miggiano ◽  
Samarpita Lahiri ◽  
Giuseppe Perugino ◽  
Maria Ciaramella ◽  
...  
Keyword(s):  
Excision Repair ◽  
Single Step ◽  
Double Stranded Dna ◽  
Nucleotide Excision ◽  
Enzymatic Systems ◽  
Methylguanine Methyltransferase ◽  
Mutagenic Effects ◽  
Wild Type Form

Mycobacterium tuberculosis (MTB) is an extremely well adapted human pathogen capable to survive for decades inside the hostile environment represented by the host's infected macrophages despite exposure to multiple potential DNA-damaging stresses. In order to maintain a remarkable low level of genetic diversity, MTB deploys different strategies of DNA repair, including multi-enzymatic systems, such as Nucleotide Excision Repair, and single-step repair. In particular, to counteract the mutagenic effects of DNA alkylation, MTB performs the direct alkylated-base reversal by sacrificing one molecule of a DNA-protein alkyltransferase, such as O6-methylguanine methyltransferase (OGT; orf: Rv1316c). We present here the biochemical and structural characterization of recombinant mycobacterial OGT (MtOGT) in its wild-type form along with its mutated variants mimicking the ones occurring in relevant clinical strains (i.e. MtOGT-T15S and MtOGT-R37L). Our studies reveal that MtOGT-R37L is severely impaired in its activity as consequence of its ten-fold lower affinity for modified double-stranded DNA (dsDNA) (1). Further investigations on a new structure-based panel of OGT versions, designed to explore different molecular environment at position 37, allowed us a better understanding of the functional role of the MtOGT Arg37-bearing loop during catalysis. Moreover, we solved the crystal structure of MtOGT in covalent complex with modified dsDNA that reveals an unprecedented MtOGT::DNA architecture, suggesting that the MtOGT monomer performing the catalysis needs assisting unreacted subunits during cooperative DNA binding. This work is supported by European Community FP7 program SYSTEMTB (Health-F4-2010-241587)

The Role of the cAMP-Response Element in the Bcl-2 Promoter in the Regulation of Endogenous Bcl-2 Expression and Apoptosis in Murine B Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2987-2987
Author(s):  
Hong Xiang ◽  
Linda M. Boxer
Keyword(s):  
B Cells ◽  
Wild Type ◽  
Camp Response Element ◽  
Response Element ◽  
Calcium Dependent ◽  
Flow Cytometric ◽  
Immature B Cells

Abstract We have previously shown in B cell lines that the cAMP-response element (CRE) is a major positive regulatory site in the bcl-2 promoter. This element is not only essential for bcl-2 deregulation in t(14;18) cells, but it is also responsible for the positive regulation of bcl-2 expression during the activation of mature B cells and the rescue of immature B cells from calcium-dependent apoptosis in vitro. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutant CRE site was introduced into the bcl-2 promoter region. The mutant CRE reduced the expression of bcl-2 mRNA in several tissues, including thymus, kidney, lung, liver, brain and heart. The levels of bcl-2 mRNA and protein were also significantly lower in splenic B cells from the knock-in mice. Consistent with these results, the activation of B cells from the knock-in mice by anti-CD 40, lipopolysaccharide (LPS) or anti-IgM was reduced as compared to B cells from wild-type littermates. B cells with the mutant CRE were more susceptible to the induction of apoptosis with several different agents consistent with the decreased expression of bcl-2. Preliminary flow cytometric studies suggest that the number of B cells is decreased in the knock-in mice at 8 weeks of age. Quantitative chromatin immunoprecipitation assays revealed essentially no binding of CREB or ATF-2 and decreased binding of CBP and c-Rel to the mutant CRE site in the bcl-2 promoter. Our previous studies have shown that the CRE site in the bcl-2 promoter is linked to the mediation of signal transduction pathways in B cells, so we investigated the effect of forskolin, a cAMP-elevating agent. We found that treatment of the B cells from the knock-in mice with forskolin led to significantly more cell death than observed with wild-type B cells. Taken together, these findings indicate that the CRE site in the bcl-2 promoter has a functional role in the regulation of endogenous bcl-2 expression and plays an important role in the regulation of apoptosis in B cells.

scholarly journals H2AX Is Required for Recombination Between Immunoglobulin Switch Regions but Not for Intra-Switch Region Recombination or Somatic Hypermutation

2003 ◽  
Vol 197 (12) ◽  
pp. 1767-1778 ◽  
Author(s):  
Bernardo Reina-San-Martin ◽  
Simone Difilippantonio ◽  
Leif Hanitsch ◽  
Revati F. Masilamani ◽  
André Nussenzweig ◽  
...  
Keyword(s):  
B Cells ◽  
Posttranslational Modifications ◽  
Somatic Hypermutation ◽  
Dna Lesions ◽  
Switch Region ◽  
Histone H2ax ◽  
Class Switch ◽  
Switch Regions ◽  
Precise Role

Changes in chromatin structure induced by posttranslational modifications of histones are important regulators of genomic function. Phosphorylation of histone H2AX promotes DNA repair and helps maintain genomic stability. Although B cells lacking H2AX show impaired class switch recombination (CSR), the precise role of H2AX in CSR and somatic hypermutation (SHM) has not been defined. We show that H2AX is not required for SHM, suggesting that the processing of DNA lesions leading to SHM is fundamentally different from CSR. Impaired CSR in H2AX−/− B cells is not due to alterations in switch region transcription, accessibility, or aberrant joining. In the absence of H2AX, short-range intra-switch region recombination proceeds normally while long-range inter-switch region recombination is impaired. Our results suggest a role for H2AX in regulating the higher order chromatin remodeling that facilitates switch region synapsis.

Bacterial DNA repair genes and their eukaryotic homologues: 4. The role of nucleotide excision DNA repair (NER) system in mammalian cells.

2007 ◽  
Vol 54 (3) ◽  
pp. 469-482 ◽  
Author(s):  
Leena Maddukuri ◽  
Dominika Dudzińska ◽  
Barbara Tudek
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Wide Spectrum ◽  
Genetic Disorders ◽  
Dna Lesions ◽  
Entire Genome ◽  
Sequence Of Events ◽  
Nucleotide Excision

The eukaryotic cell encounters more than one million various kinds of DNA lesions per day. The nucleotide excision repair (NER) pathway is one of the most important repair mechanisms that removes a wide spectrum of different DNA lesions. NER operates through two sub pathways: global genome repair (GGR) and transcription-coupled repair (TCR). GGR repairs the DNA damage throughout the entire genome and is initiated by the HR23B/XPC complex, while the CSB protein-governed TCR process removes DNA lesions from the actively transcribed strand. The sequence of events and the role of particular NER proteins are currently being extensively discussed. NER proteins also participate in other cellular processes like replication, transcription, chromatin maintenance and protein turnover. Defects in NER underlay severe genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD).

The Ets-1 transcription factor is required for Stat1-mediated T-bet expression and IgG2a class switching in mouse B cells

Blood ◽  
2012 ◽  
Vol 119 (18) ◽  
pp. 4174-4181 ◽  
Author(s):  
Hai Vu Nguyen ◽  
Enguerran Mouly ◽  
Karine Chemin ◽  
Romain Luinaud ◽  
Raymonde Despres ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
Molecular Mechanisms ◽  
Wild Type ◽  
Transcriptional Enhancer ◽  
T Box ◽  
Class Switch ◽  
Ifn Γ

Abstract In response to antigens and cytokines, mouse B cells undergo class-switch recombination (CSR) and differentiate into Ig-secreting cells. T-bet, a T-box transcription factor that is up-regulated in lymphocytes by IFN-γ or IL-27, was shown to regulate CSR to IgG2a after T cell–independent B-cell stimulations. However, the molecular mechanisms controlling this process remain unclear. In the present study, we show that inactivation of the Ets-1 transcription factor results in a severe decrease in IgG2a secretion in vivo and in vitro. No T-bet expression was observed in Ets-1–deficient (Ets-1−/−) B cells stimulated with IFN-γ and lipopolysaccharide, and forced expression of T-bet in these cells rescued IgG2a secretion. Furthermore, we identified a transcriptional enhancer in the T-bet locus with an activity in B cells that relies on ETS-binding sites. After IFN-γ stimulation of Ets-1−/− B cells, activated Stat1, which forms a complex with Ets-1 in wild-type cells, no longer binds to the T-bet enhancer or promotes histone modifications at this site. These results demonstrate that Ets-1 is critical for IgG2a CSR and acts as an essential cofactor for Stat1 in the regulation of T-bet expression in B cells.

scholarly journals Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching

2008 ◽  
Vol 205 (7) ◽  
pp. 1567-1572 ◽  
Author(s):  
Ali A. Zarrin ◽  
Peter H. Goff ◽  
Kate Senger ◽  
Frederick W. Alt
Keyword(s):  
B Cells ◽  
Heavy Chain ◽  
Recombination Event ◽  
Class Switch Recombination ◽  
Constant Region ◽  
Wild Type ◽  
Class Switch ◽  
Specific Factors ◽  
Antibody Class

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.

scholarly journals PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1

2012 ◽  
Vol 199 (2) ◽  
pp. 235-249 ◽  
Author(s):  
Alex Pines ◽  
Mischa G. Vrouwe ◽  
Jurgen A. Marteijn ◽  
Dimitris Typas ◽  
Martijn S. Luijsterburg ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Chromatin Remodeling ◽  
Excision Repair ◽  
Dna Lesions ◽  
Subsequent Removal ◽  
Wd40 Repeat ◽  
Nucleotide Excision ◽  
Efficient Recognition

The WD40-repeat protein DDB2 is essential for efficient recognition and subsequent removal of ultraviolet (UV)-induced DNA lesions by nucleotide excision repair (NER). However, how DDB2 promotes NER in chromatin is poorly understood. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a novel DDB2-associated factor. We demonstrate that DDB2 facilitated poly(ADP-ribosyl)ation of UV-damaged chromatin through the activity of PARP1, resulting in the recruitment of the chromatin-remodeling enzyme ALC1. Depletion of ALC1 rendered cells sensitive to UV and impaired repair of UV-induced DNA lesions. Additionally, DDB2 itself was targeted by poly(ADP-ribosyl)ation, resulting in increased protein stability and a prolonged chromatin retention time. Our in vitro and in vivo data support a model in which poly(ADP-ribosyl)ation of DDB2 suppresses DDB2 ubiquitylation and outline a molecular mechanism for PARP1-mediated regulation of NER through DDB2 stabilization and recruitment of the chromatin remodeler ALC1.

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