Ulf Grawunder

Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells

Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be. Here we show that DNA ligase IV (ref. 5) co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4–DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.

Yeast DNA ligase IV mediates non-homologous DNA end joining

The discovery of homologues from the yeast Saccharomyces cerevisiae of the human Ku DNA-end-binding proteins (HDF1 and KU80) has established that this organism is capable of non-homologous double-strand end joining (NHEJ), a form of DNA double-strand break repair (DSBR) active in mammalian V(D)J recombination. Identification of the DNA ligase that mediates NHEJ in yeast will help elucidate the function of the four mammalian DNA ligases in DSBR, V(D)J recombination and other reactions. Here we show that S. cerevisiae has two typical DNA ligases, the known DNA ligase I homologue CDC9 (refs 11,12, 13, 14) and the previously unknown DNA ligase IV homologue DNL4. dnl4 mutants are deficient in precise and end-processed NHEJ. DNL4 and HDF1 are epistatic in this regard, with the mutation of each having equivalent effects. dn14 mutants are complemented by overexpression of Dnl4 but not of Cdc9, and deficiency of Dnl4 alone does not impair either cell growth or the Cdc9-mediated responses to ionizing and ultraviolet radiation. Thus, S.cerevisiae has two distinct and separate ligation pathways.

DNA Ligase IV Is Essential for V(D)J Recombination and DNA Double-Strand Break Repair in Human Precursor Lymphocytes

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.

REQUIREMENT FOR AN INTERACTION OF XRCC4 WITH DNA LIGASE IV FOR WILD-TYPE V(D)J RECOMBINATION AND DNA DOUBLE-STRAND BREAK REPAIR IN VIVO

The XRCC4 gene is required for the repair of DNA double-strand breaks in mammalian cells. Without XRCC4, cells are hypersensitive to ionizing radiation and deficient for V(D)J recombination. It has been demonstrated that XRCC4 binds and stimulates DNA ligase IV, which has led to the hypothesis that DNA ligase IV is essential for both of these processes. In this study deletion mutants of XRCC4 were tested for their ability to associate with DNA ligase IVin vitro and for their ability to reconstitute XRCC4-deficient cells in vivo. We find that a central region of XRCC4 from amino acids 100–250 is necessary for DNA ligase IV binding and that deletions within this region functionally inactivates XRCC4. Deletions within the C-terminal 84 amino acids neither affect DNA ligase IV binding nor the in vivofunction of XRCC4. The correlation between the ability or inability of XRCC4 to bind DNA ligase IV and its ability or failure to reconstitute wild-type DNA repair in vivo, respectively, demonstrates for the first time that the physical interaction with DNA ligase IV is crucial for the in vivo function of XRCC4. Deletions within the N-terminal 100 amino acids inactivate XRCC4 in vivo but leave DNA ligase IV binding unaffected. This indicates further DNA ligase IV-independent functions of XRCC4.

Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks

A convergence of information from biochemistry, yeast and mammalian genetics, immunology, and radiation biology has permitted identification of some of the protein participants - Ku, DNA-PK, XRCC4 - and the reaction intermediates in DNA end joining, suggesting how broken chromosomal ends may be recognized and repaired in eukaryotic cells. Some components may be defective in inherited disorders.

The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts

There are two types of chromosome instability, structural and numerical, and these are important in cancer. Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks. Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks. NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle [1]. Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway. We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks. Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks. Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks. Rather the pre-B cells showed increased cell death. These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].

ANTIGEN RECEPTOR GENE REARRANGEMENT

Two specialized forms of site-directed double-strand (ds) DNA breakage and rejoining are part of the physiologic program of lymphocytes. One is recombination of the V, D and J gene sequences, termed V(D)J recombination, occurring during early B- and T-cell development, and the other is class-switch recombination occurring exclusively in mature B cells. For V(D)J recombination significant progress has been made recently elucidating the biochemistry of the reaction. In particular our understanding of how DNA ds breaks are both generated and rejoined has increased. For class-switch recombination no definitive information is known about the nucleases required for making the ds breaks, but recent evidence suggests that the joining phase shares activities also required for V(D)J recombination and general DNA ds break repair.

Sequence and base modifications of two phenylalanine-tRNAs from Thermus thermophilus HB8

Bulk tRNAs were prepared (1) from Thermus themophilus HB8 grown at 70°C, and purified by chromatography on BD Cellulose, Sepharose 4B, and RPC-5 (2-4). The phenylalanineaccepting tRNAs were identified by amino-acylation. Nucleotide sequences were determined chemically, by mobility shift, and by a post-labeling method (summarized in 5). HPLC (6) was used for the quantification of modified nucleosides and identification of A4 and A5 (for abbreviations of modified bases, see 7).

A novel molecular complex expressed on immature B cells: a possible role in T cell-independent B cell development.

To identify surface molecules that may play a role in regulating ileal Peyers patch (PP) B cell growth, we generated monoclonal antibodies (mAbs) and then selected them for a unique reactivity with ileal PP B cells. Flow cytometric analysis identified a mAb (SIC4.8R) that labeled 97% of ileal and 50–60% of jejunal PP sIgM+B cells. SIC4.8R also labeled a subpopulation of cortical thymocytes but few B or T cells in other lymphoid tissues, including bone marrow. Immunohistochemistry revealed intense SIC4.8R staining of B cells in the cortex of ileal PP follicles. SIC4.8R also labeled bovine PP B cells, a murine pro-B cell line, and pre-B cells in human bone marrow. Protein chemistry revealed that a structurally similar molecular complex was expressed on sheep ileal PP B cells and thymocytes and murine pro-B cells. Addition of soluble SIC4.8R to cultured ileal PP B cells reduced apoptotic cell death, elevated proliferative responses, partially inhibited anti-Ig-induced cell death, and induced IL-4 responsiveness. In contrast, soluble SIC4.8R had an antiproliferative effect on a mouse pro-B cell line. Finally, SIC4.8R labeling declined following the stimulation of ileal PP B cells with CD40 ligand. In conclusion, the present investigation determined that SIC4.8R identified a novel molecular complex that is expressed at several stages of T cell-independent B cell development in a variety of mammalian species. This observation confirmed that PP B cells are developmentally distinct from other B cell populations in sheep and suggested that the bone marrow may not be a site of B lymphopoiesis in young lambs.