Jayanta Chaudhuri

A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

Interplay of p53 and DNA-repair protein xrcc4 in tumorigenesis, genomic stability and development

XRCC4 is a non-homologous end-joining protein employed in DNA double strand break repair and in V(D)J recombination. In mice, XRCC4-deficiency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis. Here we show that p53-deficiency rescues several aspects of the XRCC4-deficient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no significant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-deficiency allowed postnatal survival of XRCC4-deficient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-deficient embryonic fibroblasts exhibited marked genomic instability including chromosomal translocations. Our findings support a crucial role for the non-homologous end-joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.

Class-switch recombination: interplay of transcription, DNA deamination and DNA repair

Class-switch recombination (CSR) of immunoglobulin heavy chains is the genetic process by which a B cell switches from the production of IgM to the production of IgG, IgE or IgA. Although the general characteristics of CSR have been known for some time, the detailed molecular mechanism of this process is only now emerging. CSR is unique, in that it seems to involve transcription-generated, higher-order RNA–DNA structures, specific DNA deamination and several DNA-repair pathways. In this review, we discuss our current knowledge of the mechanism of CSR and highlight the important unanswered questions.

A Targeted DNA-PKcs-Null Mutation Reveals DNA-PK-Independent Functions for KU in V(D)J Recombination

The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.

Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation

Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the ‘capture’ or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc−/− mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.

Leaky Scid Phenotype Associated with Defective V(D)J Coding End Processing in Artemis-Deficient Mice

Radiosensitive severe combined immune deficiency in humans results from mutations in Artemis, a protein which, when coupled with DNA-dependent protein kinase catalytic subunit (DNA-PKcs), possesses DNA hairpin-opening activity in vitro. Here, we report that Artemis-deficient mice have an overall phenotype similar to that of DNA-PKcs-deficient mice-including severe combined immunodeficiency associated with defects in opening and joining V(D)J coding hairpin ends and increased cellular ionizing radiation sensitivity. While these findings strongly support the notion that Artemis functions with DNA-PKcs in a subset of NHEJ functions, differences between Artemis- and DNA-PKcs-deficient phenotypes, most notably decreased fidelity of V(D)J signal sequence joining in DNA-PKcs-deficient but not Artemis-deficient fibroblasts, suggest additional functions for DNA-PKcs. Finally, Artemis deficiency leads to chromosomal instability in fibroblasts, demonstrating that Artemis functions as a genomic caretaker.

Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.

V(D)J recombination is the process by which the variable region exons encoding the antigen recognition sites of receptors expressed on B and T lymphocytes are generated during early development via somatic assembly of component gene segments. In response to antigen, somatic hypermutation (SHM) and class switch recombination (CSR) induce further modifications of immunoglobulin genes in B cells. CSR changes the IgH constant region for an alternate set that confers distinct antibody effector functions. SHM introduces mutations, at a high rate, into variable region exons, ultimately allowing affinity maturation. All of these genomic alteration processes require tight regulatory control mechanisms, both to ensure development of a normal immune system and to prevent potentially oncogenic processes, such as translocations, caused by errors in the recombination/mutation processes. In this regard, transcription of substrate sequences plays a significant role in target specificity, and transcription is mechanistically coupled to CSR and SHM. However, there are many mechanistic differences in these reactions. V(D)J recombination proceeds via precise DNA cleavage initiated by the RAG proteins at short conserved signal sequences, whereas CSR and SHM are initiated over large target regions via activation-induced cytidine deaminase (AID)-mediated DNA deamination of transcribed target DNA. Yet, new evidence suggests that AID cofactors may help provide an additional layer of specificity for both SHM and CSR. Whereas repair of RAG-induced double-strand breaks (DSBs) involves the general nonhomologous end-joining DNA repair pathway, and CSR also depends on at least some of these factors, CSR requires induction of certain general DSB response factors, whereas V(D)J recombination does not. In this review, we compare and contrast V(D)J recombination and CSR, with particular emphasis on the role of the initiating enzymes and DNA repair proteins in these processes.

The role of the non-homologous end-joining pathway in lymphocyte development.

Summary:  One of the most toxic insults a cell can incur is a disruption of its linear DNA in the form of a double‐strand break (DSB). Left unrepaired, or repaired improperly, these lesions can result in cell death or neoplastic transformation. Despite these dangers, lymphoid cells purposely introduce DSBs into their genome to maximize the diversity and effector functions of their antigen receptor genes. While the generation of breaks requires distinct lymphoid‐specific factors, their resolution requires various ubiquitously expressed DNA‐repair proteins, known collectively as the non‐homologous end‐joining pathway. In this review, we discuss the factors that constitute this pathway as well as the evidence of their involvement in two lymphoid‐specific DNA recombination events.

An evolutionarily conserved target motif for immunoglobulin class-switch recombination

Immunoglobulin H class-switch recombination (CSR) occurs between switch regions and requires transcription and activation-induced cytidine deaminase (AID). Transcription through mammalian switch regions, because of their GC-rich composition, generates stable R-loops, which provide single-stranded DNA substrates for AID. However, we show here that the Xenopus laevis switch region Sμ, which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate CSR in vivo. X. laevis Sμ–mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID–replication protein A complex when transcribed in vitro. We propose that AGCT is a primordial CSR motif that targets AID through a non-R-loop mechanism involving an AID–replication protein A complex.