Chengming Zhu

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.

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway.

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.

Ku86-Deficient Mice Exhibit Severe Combined Immunodeficiency and Defective Processing of V(D)J Recombination Intermediates

Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining.

Growth retardation and leaky SCID phenotype of Ku70-deficient mice

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations.

Amplification of large genomic regions associated with complex translocations (complicons) is a basis for tumor progression and drug resistance. We show that pro-B lymphomas in mice deficient for both p53 and nonhomologous end-joining (NHEJ) contain complicons that coamplify c-myc (chromosome 15) and IgH (chromosome 12) sequences. While all carry a translocated (12;15) chromosome, coamplified sequences are located within a separate complicon that often involves a third chromosome. Complicon formation is initiated by recombination of RAG1/2-catalyzed IgH locus double-strand breaks with sequences downstream of c-myc, generating a dicentric (15;12) chromosome as an amplification intermediate. This recombination event employs a microhomology-based end-joining repair pathway, as opposed to classic NHEJ or homologous recombination. These findings suggest a general model for oncogenic complicon formation.

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.

V(D)J Recombination in Ku86-Deficient Mice: Distinct Effects on Coding, Signal, and Hybrid Joint Formation

Ku, a heterodimer of 70 and 86 kDa subunits, plays a critical but poorly understood role in V(D)J recombination. Although Ku86-deficient mice are defective in coding and signal joint formation, rare recombination products have been detected by PCR. Here, we report nucleotide sequences of 99 junctions from Ku86-deficient mice. Over 90% of the coding joints, but not signal or hybrid joints, exhibit short sequence homologies, indicating that homology is required to join coding ends in the absence of Ku86. Our results suggest that Ku86 may normally have distinct functions in the formation of these different types of junctions. Furthermore, Ku86(-/-) joints are unexpectedly devoid of N-region diversity, suggesting a novel role for Ku in the addition of N nucleotides by terminal deoxynucleotidyl transferase.

Characterization of coding ends in thymocytes of scid mice: Implications for the mechanism of V(D)J recombination

We previously identified possible intermediates in V(D)J recombination at the TCR delta locus and characterized molecules with signal ends and with covalently sealed (hairpin) coding ends in thymocytes of scid mice by Southern blotting. Here, we use a sensitive ligation-mediated PCR assay to demonstrate that all coding ends detected in scid thymocytes are covalently sealed. Neither coding nor signal ends exhibit loss or addition of nucleotides. These data imply that hairpin formation is coupled to the initial cleavage at the signal/coding border, and that the cleavage step in V(D)J recombination is conservative. In scid/+ or wild-type thymocytes, hairpin coding ends are at least 1000-fold less abundant than signal ends. These results provide insight into the mechanism of V(D)J recombination.