Yunmei Ma

Mechanism and regulation of human non-homologous DNA end-joining

Non-homologous DNA end-joining (NHEJ) — the main pathway for repairing double-stranded DNA breaks — functions throughout the cell cycle to repair such lesions. Defects in NHEJ result in marked sensitivity to ionizing radiation and ablation of lymphocytes, which rely on NHEJ to complete the rearrangement of antigen-receptor genes. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes, but which might also contribute to some of the genetic changes that underlie cancer and ageing.

Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV

DNA double‐strand breaks (dsb) during V(D)J recombination of T and B lymphocyte receptor genes are resolved by the non‐homologous DNA end joining pathway (NHEJ) including at least six factors: Ku70, Ku80, DNA‐PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Artemis and Lig4 are the only known V(D)J/NHEJ factors found deficient in human genetic disorders. Null mutations of the Artemis gene result in a complete absence of T and B lymphocytes and increased cellular sensitivity to ionizing radiations, causing radiosensitive‐SCID. Mutations of Lig4 are exclusively hypomorphic and have only been described in six patients, four exhibiting mild immunodeficiency associated with microcephaly and developmental delay, while two patient had leukemia. Here we report a SCID associated with microcephaly caused by compound heterozygous hypomorphic mutations in Lig4. Residual activity of Lig4 in these patients is underscored by a normal pattern of TCR‐α and ‐β junctions in the T cells of the patients and a moderate impairment of V(D)J recombination as tested in vitro. These observations contrast with the severity of the clinical immunodeficiency, suggesting that Lig4 may have additional critical roles in lymphocyte survival beyond V(D)J recombination.

The DNA-dependent Protein Kinase Catalytic Subunit Phosphorylation Sites in Human Artemis

Artemis protein has irreplaceable functions in V(D)J recombination and nonhomologous end joining (NHEJ) as a hairpin and 5′ and 3′ overhang endonuclease. The kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is necessary in activating Artemis as an endonuclease. Here we report that three basal phosphorylation sites and 11 DNA-PKcs phosphorylation sites within the mammalian Artemis are all located in the C-terminal domain. All but one of these phosphorylation sites deviate from the SQ or TQ motif of DNA-PKcs that was predicted previously from in vitro phosphorylation studies. Phosphatase-treated mammalian Artemis and Artemis that is mutated at the three basal phosphorylation sites still retain DNA-PKcs-dependent endonucleolytic activities, indicating that basal phosphorylation is not required for the activation. In vivo studies of Artemis lacking the C-terminal domain have been reported to be sufficient to complement V(D)J recombination in Artemis null cells. Therefore, the C-terminal domain may have a negative regulatory effect on the Artemis endonucleolytic activities, and phosphorylation by DNA-PKcs in the C-terminal domain may relieve this inhibition.

Functional and biochemical dissection of the structure-specific nuclease ARTEMIS

During V(D)J recombination, the RAG1 and RAG2 proteins form a complex and initiate the process of rearrangement by cleaving between the coding and signal segments and generating hairpins at the coding ends. Prior to ligation of the coding ends by DNA ligase IV/XRCC4, these hairpins are opened by the ARTEMIS/DNA‐PKcs complex. ARTEMIS, a member of the metallo‐β‐lactamase superfamily, shares several features with other family members that act on nucleic acids. ARTEMIS exhibits exonuclease and, in concert with DNA‐PKcs, endonuclease activities. To characterize amino acids essential for its catalytic activities, we mutated nine evolutionary conserved histidine and aspartic acid residues within ARTEMIS. Biochemical analyses and a novel in vivo V(D)J recombination assay allowed the identification of eight mutants that were defective in both overhang endonucleolytic and hairpin‐opening activities; the 5′ to 3′ exonuclease activity of ARTEMIS, however, was not impaired by these mutations. These results indicate that the hairpin‐opening activity of ARTEMIS and/or its overhang endonucleolytic activity are necessary but its exonuclease activity is not sufficient for the process of V(D)J recombination.

Repair of double-strand DNA breaks by the human nonhomologous DNA end joining pathway: the iterative processing model.

Naturally-occurring ionizing radiation and reactive oxygen species (ROS) from oxidative metabolism are factors that have challenged all life forms during the course of evolution. Ionizing radiation (IR) and reactive oxygen species cause a diverse set of double-strand DNA end configurations. Nonhomologous DNA end joining (NHEJ) is an optimal DNA repair pathway for dealing with such a diverse set of DNA lesions. NHEJ can carry out nucleolytic, polymerase, and ligation operations on each strand independently. This iterative processing nature of NHEJ is ideal for repair of pathologic and physiologic double-strand breaks because it permits sequential action of the NHEJ enzymes on each DNA end and on each strand. The versatility of the Artemis:DNA-PKcs endonuclease in cleaving 5’ and 3’ overhangs, hairpins, gaps, flaps, and various loop conformations makes it well-suited for DNA end modifications on oxidized overhangs. In addition, the ability to cleave stem-loop and hairpin structures permits it to open terminal fold-back configurations that may arise at DNA ends after IR damage. The ability of the XRCC4:DNA ligase IV complex to ligate one strand without ligation of the other permits additional end joining flexibility in NHEJ and raises the possibility of optional involvement of repair proteins from other pathways.

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5′ and 3′ overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5′ and 3′ overhang substrates for the endonucleolytic function of Artemis.

Double-Strand Break Formation by the RAG Complex at the Bcl-2 Major Breakpoint Region and at Other Non-B DNA Structures In Vitro

ABSTRACT The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs.

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PKcs, to that end. The Ku·DNA-PKcs complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP6) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP6 bound to DNA-PKcs (not to Ku). Here we clearly show that, in fact, IP6 associates not with DNA-PKcs, but rather with Ku. Furthermore, the binding of DNA ends and IP6 to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.

In vitro nonhomologous DNA end joining system

The nonhomologous end joining (NHEJ) pathway is the major pathway that repairs DNA double strand breaks in multicellular eukaryotic organisms. Unlike homologous recombination, the NHEJ pathway utilizes minimal or no homology between the ends that need to be joined. Although the resulting NHEJ-repaired junctions can be diverse in sequence, they share a few common features, including frequent nucleolytic resection of the ends, near-random junctional additions, and utilization of microhomology. The in vitro NHEJ assay was developed in an attempt to recapitulate the joining of incompatible ends with purified core proteins and some additional factors. This in vitro system allows further understanding of the biochemical features of the pathway and evaluation of the functions of other proteins in NHEJ.