scholarly journals Similar Effects of Brca2 Truncation and Rad51 Paralog Deficiency on Immunoglobulin V Gene Diversification in DT40 Cells Support an Early Role for Rad51 Paralogs in Homologous Recombination

2005 ◽  
Vol 25 (3) ◽  
pp. 1124-1134 ◽  
Author(s):  
Atsushi Hatanaka ◽  
Mitsuyoshi Yamazoe ◽  
Julian E. Sale ◽  
Minoru Takata ◽  
Kazuhiko Yamamoto ◽  
...  
Keyword(s):  
Gene Segment ◽  
Dna Recombination ◽  
Suppressor Gene ◽  
Nucleotide Substitutions ◽  
Gene Diversification ◽  
Brca2 Protein ◽  
Rad51 Paralogs ◽  
Error Prone Repair ◽  
Variable Gene ◽  
Dt40 Cells

ABSTRACT BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3′ end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.

scholarly journals Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression

2012 ◽  
Vol 209 (12) ◽  
pp. 2199-2213 ◽  
Author(s):  
Sarah E. Powers ◽  
Malay Mandal ◽  
Satoshi Matsuda ◽  
Ana V. Miletic ◽  
Matthew H. Cato ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Gene Segment ◽  
Cyclin D3 ◽  
Cyclin D2 ◽  
Cycle Progression ◽  
Nuclear Compartment ◽  
Regulation Of Proliferation ◽  
Variable Gene ◽  
V Gene ◽  
Phosphoinositide 3 Kinase

Ubiquitously expressed D-type cyclins are required for hematopoiesis but are dispensable in other cell lineages. Furthermore, within different hematopoietic progenitor populations the D-type cyclins play nonredundant roles. The basis of this lineage and developmental specificity is unknown. In pro–B cells we demonstrate four distinct nuclear D-type cyclin compartments, including one cyclin D3 fraction associated with CDK4 and another phosphoinositide 3-kinase–regulated fraction not required for proliferation. A third fraction of cyclin D3 was associated with the nuclear matrix and repression of >200 genes including the variable (V) gene segments Igkv1-117, Iglv1, and Igh-VJ558. Consistent with different subnuclear compartments and functions, distinct domains of cyclin D3 mediated proliferation and Igk V gene segment repression. None of the cyclin D3 nuclear compartments overlapped with cyclin D2, which was distributed, unbound to CDK4, throughout the nucleus. Furthermore, compartmentalization of the cyclins appeared to be lineage restricted because in fibroblasts, cyclin D2 and cyclin D3 occupied a single nuclear compartment and neither bound CDK4 efficiently. These data suggest that subnuclear compartmentalization enables cyclin D3 to drive cell cycle progression and repress V gene accessibility, thereby ensuring coordination of proliferation with immunoglobulin recombination.

scholarly journals Cancer missense mutations in the BRC repeats of BRCA2 protein disrupt RAD51 binding and activity leading to chemotherapeutic sensitivity

2021 ◽  
Author(s):  
Judit Jimenez-Sainz ◽  
Joshua Mathew ◽  
Jennifer Garbarino ◽  
Joseph P Eder ◽  
Ryan B Jensen
Keyword(s):  
Genome Stability ◽  
Treatment Options ◽  
Suppressor Gene ◽  
Current Treatment ◽  
Missense Mutations ◽  
Dna Double Strand Breaks ◽  
Clinical Databases ◽  
Brca2 Protein ◽  
Hypomorphic Alleles ◽  
The Impact

BRCA2 is a tumor suppressor gene that maintains genome stability by mediating the high fidelity repair of DNA double-strand breaks (DSBs) through homology-directed repair (HDR). Pathogenic mutations in BRCA2 predispose to breast, ovarian, pancreatic, prostate, and other cancers. Mutations in BRCA2 leading to severe protein truncation predict pathogenicity, however, missense mutations with unknown functional consequences, designated Variants of Uncertain Significance (VUS), comprise 60% of BRCA2 sequence changes deposited in clinical databases. Classifying BRCA2 VUS correctly is critical for relaying clinically actionable information to patients concerning future cancer risk or current treatment options. In this study, we identified and biochemically characterized three BRCA2 VUS located in BRC repeats to determine the impact on canonical HDR functions. Two of the germline variants, S1221P and T1980I, map to conserved residues in BRC2 and BRC7, disrupt RAD51 binding, and are diminished in their ability to stabilize RAD51-ssDNA complexes. We provide supporting cellular evidence that S1221P and T1980I are significantly compromised in their response to chemotherapeutics and ionizing radiation. The third variant, T1346I, lies within the spacer region between BRC2 and BRC3 but remains fully functional. We conclude that T1346I has a neutral impact on BRCA2 function, while S1221P and T1980I are hypomorphic alleles that disrupt the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments.

scholarly journals Reverse genetic studies of homologous DNA recombination using the chicken B–lymphocyte line, DT40

2001 ◽  
Vol 356 (1405) ◽  
pp. 111-117 ◽  
Author(s):  
Eiichiro Sonoda ◽  
Ciaran Morrison ◽  
Yukiko M. Yamashita ◽  
Minoru Takata ◽  
Shunichi Takeda
Keyword(s):  
Cell Line ◽  
Reverse Genetics ◽  
B Lymphocyte ◽  
Dna Recombination ◽  
Temperature Sensitive ◽  
Immunoglobulin Gene ◽  
Genetic Studies ◽  
Transformed Cell Line ◽  
Variable Segment ◽  
Dt40 Cells

DT40 is an avian leucosis virus–transformed chicken B–lymphocyte line which exhibits high ratios of targeted to random integration of transfected DNA constructs. This efficient targeted integration may be related to the ongoing diversification of the variable segment of the immunoglobulin gene through homologous DNA recombination–controlled gene conversion. DT40s are a convenient model system for making gene–targeted mutants. Another advantage is the relative tractability of these cells, which makes it possible to disrupt multiple genes in a single cell and to generate conditionally gene–targeted mutants including temperature–sensitive mutants. There are strong phenotypic similarities between murine and DT40 mutants of various genes involved in DNA recombination. These similarities confirm that the DT40 cell line is a reasonable model for the analysis of vertebrate DNA recombination, despite obvious concerns associated with the use of a transformed cell line, which may have certain cell–line–specific characteristics. Here we describe our studies of homologous DNA recombination in vertebrate somatic cells using reverse genetics in DT40 cells.

Variable gene segment-specific N-insertions at the signal joint of T-cell receptor Vβ-Dβ recombinations

1998 ◽  
Vol 61 (2-3) ◽  
pp. 151-155 ◽  
Author(s):  
Yasuyoshi Kanari ◽  
Ryusuke Nakagawa ◽  
Hiroshi Arakawa ◽  
Hideo Yamagishi
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Gene Segment ◽  
Cell Receptor ◽  
Variable Gene ◽  
T Cell Receptor Vβ ◽  
Variable Gene Segment

scholarly journals Warsaw breakage syndrome DDX11 helicase acts jointly with RAD17 in the repair of bulky lesions and replication through abasic sites

2018 ◽  
Vol 115 (33) ◽  
pp. 8412-8417 ◽  
Author(s):  
Takuya Abe ◽  
Masato Ooka ◽  
Ryotaro Kawasumi ◽  
Keiji Miyata ◽  
Minoru Takata ◽  
...  
Keyword(s):  
Developmental Disorder ◽  
Genome Instability ◽  
Dna Damage Tolerance ◽  
Checkpoint Activation ◽  
Abasic Sites ◽  
Interstrand Crosslink ◽  
Recombination Repair ◽  
Variable Gene ◽  
Homeologous Recombination ◽  
Dt40 Cells

Warsaw breakage syndrome, a developmental disorder caused by mutations in the DDX11/ChlR1 helicase, shows cellular features of genome instability similar to Fanconi anemia (FA). Here we report that DDX11-deficient avian DT40 cells exhibit interstrand crosslink (ICL)-induced chromatid breakage, with DDX11 functioning as backup for the FA pathway in regard to ICL repair. Importantly, we establish that DDX11 acts jointly with the 9-1-1 checkpoint clamp and its loader, RAD17, primarily in a postreplicative fashion, to promote homologous recombination repair of bulky lesions, but is not required for intra-S checkpoint activation or efficient fork progression. Notably, we find that DDX11 also promotes diversification of the chicken Ig-variable gene, a process triggered by programmed abasic sites, by facilitating both hypermutation and homeologous recombination-mediated gene conversion. Altogether, our results uncover that DDX11 orchestrates jointly with 9-1-1 and its loader, RAD17, DNA damage tolerance at sites of bulky lesions, and endogenous abasic sites. These functions may explain the essential roles of DDX11 and its similarity with 9-1-1 during development.

scholarly journals Regulation of TCR δ and α repertoires by local and long-distance control of variable gene segment chromatin structure

2005 ◽  
Vol 202 (4) ◽  
pp. 467-472 ◽  
Author(s):  
Abbas Hawwari ◽  
Michael S. Krangel
Keyword(s):  
Chromatin Structure ◽  
Genetic Locus ◽  
Gene Segment ◽  
Chromatin Accessibility ◽  
Small Subset ◽  
Double Positive ◽  
Long Distance ◽  
Variable Gene ◽  
Distance Regulation ◽  
Variable Gene Segment

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4−CD8− (double negative [DN]) and CD4+CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of ∼100 variable gene segments contributes substantially to the adult TCRδ repertoire. Moreover, although most contribute to the TCRα repertoire, variable gene segments that are Jα proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRδ repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

scholarly journals Boundaries of somatic mutation in rearranged immunoglobulin genes: 5' boundary is near the promoter, and 3' boundary is approximately 1 kb from V(D)J gene.

1990 ◽  
Vol 172 (6) ◽  
pp. 1717-1727 ◽  
Author(s):  
S G Lebecque ◽  
P J Gearhart
Keyword(s):  
Structural Information ◽  
Gene Segment ◽  
Point Mutations ◽  
Sequence Motif ◽  
Immunoglobulin Gene ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Coding Regions ◽  
Dna Strands ◽  
Transcriptional State

To investigate why somatic mutations are spatially restricted to a region around the rearranged V(D)J immunoglobulin gene, we compared the distribution of mutations flanking murine V gene segments that had rearranged next to either proximal or distal J gene segments. 124 nucleotide substitutions, nine deletions, and two insertions were identified in 32,481 bp of DNA flanking the coding regions from 17 heavy and kappa light chain genes. Most of the mutations occurred within a 2-kb region centered around the V(D)J gene, regardless of which J gene segment was used, suggesting that the structural information for mutation is located in sequences around and within the V(D)J gene, and not in sequences downstream of the J gene segments. The majority of mutations were found within 300 bp of DNA flanking the 5' side of the V(D)J gene and 850 bp flanking the 3' side at a frequency of 0.8%, which was similar to the frequency in the coding region. The frequency of flanking mutations decreased as a function of distance from the gene. There was no evidence for hot spots in that every mutation was unique and occurred at a different position. No mutations were found upstream of the promoter region, suggesting that the promoter delimits a 5' boundary, which provides strong evidence that transcription is necessary to generate mutation. The 3' boundary was approximately 1 kb from the V(D)J gene and was not associated with a DNA sequence motif. Occasional mutations were located in the nuclear matrix association and enhancer regions. The pattern of substitutions suggests that there is discrimination between the two DNA strands during mutation, in that the four bases were mutated with different frequencies on each strand. The high frequency of mutations in the 3' flanking region and the uniqueness of each mutation argues against templated gene conversion as a mechanism for generating somatic diversity in murine V(D)J genes. Rather, the data support a model for random point mutations where the mechanism is linked to the transcriptional state of the gene.

Mouse T-cell receptor variable gene segment families

1995 ◽  
Vol 42 (6) ◽  
Author(s):  
Bernhard Arden ◽  
StephenP. Clark ◽  
Dieter Kabelitz ◽  
TakW. Mak
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Gene Segment ◽  
Cell Receptor ◽  
Variable Gene ◽  
Variable Gene Segment

scholarly journals openPrimeR for multiplex amplification of highly diverse templates

2019 ◽  
Author(s):  
Christoph Kreer ◽  
Matthias Döring ◽  
Nathalie Lehnen ◽  
Meryem S. Ercanoglu ◽  
Lutz Gieselmann ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Gene Segment ◽  
Pcr Primers ◽  
Proof Of Concept ◽  
Minimal Set ◽  
Multiplex Amplification ◽  
Variable Gene ◽  
Primer Sets ◽  
Correct Understanding ◽  
Intuitive Interface

AbstractTo study the diversity of immune receptors and pathogens, multiplex PCR has become a central approach in research and diagnostics. However, insufficient primer design against highly diverse templates often prevents amplification and therefore limits the correct understanding of biological processes. Here, we present openPrimeR, an R-based tool for evaluating and designing multiplex PCR primers. openPrimeR provides a functional and intuitive interface and uses either a greedy algorithm or an integer linear program to compute the minimal set of primers that performs full target coverage. As proof of concept, we used openPrimeR to find optimal primer sets for the amplification of highly mutated immunoglobulins. Comprehensive analyses on specifically generated immunoglobulin variable gene segment libraries resulted in the composition of highly effective primer sets (oPR-IGHV, oPR-IGKV and oPR-IGLV) that demonstrated to be particularly suitable for the isolation of novel human antibodies.

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