scholarly journals Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

1998 ◽  
Vol 18 (4) ◽  
pp. 2029-2037 ◽  
Author(s):  
Mark S. Schlissel
Keyword(s):  
Receptor Gene ◽  
Cell Receptor ◽  
Recombination Reaction ◽  
Immunoglobulin Gene ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Recombination Signal Sequences ◽  
Nuclease Digestion ◽  
Dna Ends ◽  
In Cells

ABSTRACT The V(D)J recombinase recognizes a pair of immunoglobulin or T-cell receptor gene segments flanked by recombination signal sequences and introduces double-strand breaks, generating two signal ends and two coding ends. Broken coding ends were initially identified as covalently closed hairpin DNA molecules. Before recombination, however, the hairpins must be opened and the ends must be modified by nuclease digestion and N-region addition. We have now analyzed nonhairpin coding ends associated with various immunoglobulin gene segments in cells undergoing V(D)J recombination. We found that these broken DNA ends have different nonrandom 5′-strand deletions which were characteristic for each locus examined. These deletions correlate well with the sequence characteristics of coding joints involving these gene segments. In addition, unlike broken signal ends, these nonhairpin coding-end V(D)J recombination reaction intermediates have 3′ overhanging ends. We discuss the implications of these results for models of how sequence modifications occur during coding-joint formation.

scholarly journals Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

2002 ◽  
Vol 195 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Robert E. Tillman ◽  
Andrea L. Wooley ◽  
Maureen M. Hughes ◽  
Tara D. Wehrly ◽  
Wojciech Swat ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Receptor Gene ◽  
Antigen Receptor ◽  
Double Strand Breaks ◽  
Recombination Reaction ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Signal Sequences ◽  
Dna Dsbs ◽  
Cleavage Step

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Role of RAG1 autoubiquitination in V(D)J recombination

2015 ◽  
Vol 112 (28) ◽  
pp. 8579-8583 ◽  
Author(s):  
Samarendra K. Singh ◽  
Martin Gellert
Keyword(s):  
Dna Cleavage ◽  
E3 Ligase ◽  
Cell Receptor ◽  
Recombination Reaction ◽  
Neighboring Gene ◽  
Ubiquitin E3 Ligase ◽  
Recombination Signal Sequences ◽  
Ubiquitin Ligase Activity ◽  
Variable Domains ◽  
Mouse Cells

The variable domains of Ig and T-cell receptor genes in vertebrates are assembled from gene fragments by the V(D)J recombination process. The RAG1–RAG2 recombinase (RAG1/2) initiates this recombination by cutting DNA at the borders of recombination signal sequences (RSS) and their neighboring gene segments. The RAG1 protein is also known to contain a ubiquitin E3 ligase activity, located in an N-terminal region that is not strictly required for the basic recombination reaction but helps to regulate recombination. The isolated E3 ligase domain was earlier shown to ubiquitinate one site in a neighboring RAG1 sequence. Here we show that autoubiquitination of full-length RAG1 at this specific residue (K233) results in a large increase of DNA cleavage by RAG1/2. A mutational block of the ubiquitination site abolishes this effect and inhibits recombination of a test substrate in mouse cells. Thus, ubiquitination of RAG1, which can be promoted by RAG1’s own ubiquitin ligase activity, plays a significant role in governing the level of V(D)J recombination activity.

Diagnosis of Canine Lymphoid Neoplasia Using Clonal Rearrangements of Antigen Receptor Genes

2003 ◽  
Vol 40 (1) ◽  
pp. 32-41 ◽  
Author(s):  
R. C. Burnett ◽  
W. Vernau ◽  
J. F. Modiano ◽  
C. S. Olver ◽  
P. F. Moore ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Gene Rearrangement ◽  
Receptor Gene ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Immunoglobulin Gene ◽  
Lymphoid Malignancy ◽  
Variable Regions ◽  
Lymphoid Neoplasia

Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1–10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.

scholarly journals Evidence for peripheral blood B lymphocyte but not T lymphocyte involvement in multiple myeloma

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1550-1553 ◽  
Author(s):  
J Berenson ◽  
R Wong ◽  
K Kim ◽  
N Brown ◽  
A Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Receptor Gene ◽  
Cell Receptor ◽  
Peripheral Blood Lymphocytes ◽  
Immunoglobulin Gene

Because there is controversy regarding whether subsets of peripheral blood lymphocytes (PBLs) are part of the malignant clone in patients with multiple myeloma, we studied this question by immunoglobulin and T cell receptor gene analysis. Southern blot analysis with antibody probes demonstrated clonal immunoglobulin gene rearrangements in PBLs of seven of nine patients that were identical to those seen in their marrow plasma cells. Circulating plasma cells were not detected in any of these patients. In contrast, no patient demonstrated clonally rearranged T cell receptor genes. In one sequentially studied patient, PBLs obtained at diagnosis when he had stage I (Durie-Salmon) contained only germline DNA, while analysis of PBLs at relapse (stage III) revealed a clonally rearranged band. These data confirm the notion that circulating lymphocytes in patients with myeloma are part of the malignant clone and, furthermore, these malignant cells are of B cell rather than T cell lineage.

Loss of juxtaposition of RAG-induced immunoglobulin DNA ends is implicated in the precursor B-cell differentiation defect in NBS patients

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4770-4777 ◽  
Author(s):  
Mirjam van der Burg ◽  
Malgorzata Pac ◽  
Magdalena A. Berkowska ◽  
Bozenna Goryluk-Kozakiewicz ◽  
Anna Wakulinska ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Gene Segment ◽  
Cell Receptor ◽  
Nijmegen Breakage Syndrome ◽  
Immunoglobulin Gene ◽  
B Cell Differentiation ◽  
Receptor Repertoire ◽  
Recombination Activating Gene ◽  
Dna Ends

Abstract The Nijmegen breakage syndrome (NBS) is a rare inherited condition, characterized by microcephaly, radiation hypersensitivity, chromosomal instability, an increased incidence of (mostly) lymphoid malignancies, and immunodeficiency. NBS is caused by hypomorphic mutations in the NBN gene (8q21). The NBN protein is a subunit of the MRN (Mre11-Rad50-NBN) nuclear protein complex, which associates with double-strand breaks. The immunodeficiency in NBS patients can partly be explained by strongly reduced absolute numbers of B lymphocytes and T lymphocytes. We show that NBS patients have a disturbed precursor B-cell differentiation pattern and significant disturbances in the resolution of recombination activating gene-induced IGH breaks. However, the composition of the junctional regions as well as the gene segment usage of the reduced number of successful immunoglobulin gene rearrangements were highly similar to healthy controls. This indicates that the NBN defect leads to a quantitative defect in V(D)J recombination through loss of juxtaposition of recombination activating gene-induced DNA ends. The resulting reduction in bone marrow B-cell efflux appeared to be partly compensated by significantly increased proliferation of mature B cells. Based on these observations, we conclude that the quantitative defect will affect the B-cell receptor repertoire, thus contributing to the observed immunodeficiency in NBS patients.

scholarly journals Role for RAD18 in Homologous Recombination in DT40 Cells

2006 ◽  
Vol 26 (21) ◽  
pp. 8032-8041 ◽  
Author(s):  
Dávid Szüts ◽  
Laura J. Simpson ◽  
Sarah Kabani ◽  
Mitsuyoshi Yamazoe ◽  
Julian E. Sale
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Early Stage ◽  
Current Evidence ◽  
Recombination Reaction ◽  
Immunoglobulin Gene ◽  
Postreplication Repair ◽  
Strand Breaks ◽  
Dt40 Cells

ABSTRACT RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

scholarly journals Positive and Negative Regulation of  V(D)J Recombination by the E2A Proteins

1999 ◽  
Vol 189 (2) ◽  
pp. 289-300 ◽  
Author(s):  
Gretchen Bain ◽  
William J. Romanow ◽  
Karen Albers ◽  
Wendy L. Havran ◽  
Cornelis Murre
Keyword(s):  
Adult Development ◽  
Signal Sequence ◽  
Gene Segment ◽  
Cell Receptor ◽  
Recombination Reaction ◽  
Gene Products ◽  
Antigen Receptors ◽  
Dna Breaks ◽  
Thymocyte Development ◽  
Temporal Regulation

A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) γ and δ loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the γ and δ V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vγ3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vγ3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vγ3. Additionally, rearrangements to a number of Vγ and Vδ gene segments used predominately during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vδ gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vδ regions.

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