Howard H. Y. Chang

Non-homologous DNA end joining and alternative pathways to double-strand break repair

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Significance Nonhomologous end-joining (NHEJ) is the main pathway for repair of DNA double-strand breaks (DSBs), the most cytotoxic form of DNA damage resulting from ionizing radiation, chemotherapeutics, and normal cellular processes. The mechanisms that control NHEJ play key roles in development, in immunity, and in response to cancer therapy; however, the current state of knowledge regarding the physical nature of the NHEJ repair process is limited. Here we used super-resolution microscopy to define the organization of NHEJ complexes in cells, showing that long filaments form at either side of the break. Single-molecule FRET revealed dynamic behavior in which breaks can pair in an adjacent, non–end-to-end configuration. Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

Role of the Aspartyl-Asparaginyl-β-Hydroxylase Gene in Neuroblastoma Cell Motility

Aspartyl (asparaginyl) β-hydroxylase (AAH) is overexpressed in various malignant neoplasms, and high levels of immunoreactivity mainly occur in infiltrating or metastasized tumors. In addition, AAH is abundantly expressed in normally invasive placental trophoblastic cells. These observations led to the hypothesis that AAH may have a role in motility and aggressive behavior of tumor cells. The present study demonstrates that AAH is overexpressed in primary human malignant neuroectodermal tumors, including medulloblastomas and neuroblastomas, and that AAH expression is at a low level or undetectable in the normal mature brain. In the Sy5y neuroblastoma cell line, endogenous expression of the ∼86-kd AAH protein was demonstrated by Western blot analysis, and immunoreactivity predominantly localized to the cell surface by immunocytochemical staining and FACS analysis. Sy5y cells that were stably transfected with the human AAH cDNA had increased levels of proliferating cell nuclear antigen and Bcl-2, and reduced levels of p21/Waf1 and p16. In addition, increased AAH expression enhanced Sy5y cell motility, whereas antisense oligodeoxynucleotide inhibition of AAH significantly reduced Sy5y cell motility and increased the levels of p21/Waf1 and p16. The findings suggest that AAH overexpression contributes to the malignant phenotype of neuroectodermal tumor cells by increasing motility and enhancing proliferation, survival, and cell cycle progression. Because AAH expression is at a low level or undetectable in normal brain, the AAH gene may be a target for treating primitive neuroectodermal tumors.

Evidence that the DNA endonuclease ARTEMIS also has intrinsic 5'-exonuclease activity.

Background: DNA-PKcs stimulates the endonucleolytic activities of ARTEMIS but not the 5′-exonuclease activity. Results: Point mutagenesis, small molecule inhibitors, and modulation of divalent cation concentrations all affect both the endonuclease and 5′-exonuclease activities in parallel. Conclusion: ARTEMIS has intrinsic 5′-exonuclease activity, in addition to its known endonuclease activity. Significance: ARTEMIS may use both its endo- and its 5′-exonuclease activity in NHEJ. ARTEMIS is a member of the metallo-β-lactamase protein family. ARTEMIS has endonuclease activity at DNA hairpins and at 5′- and 3′-DNA overhangs of duplex DNA, and this endonucleolytic activity is dependent upon DNA-PKcs. There has been uncertainty about whether ARTEMIS also has 5′-exonuclease activity on single-stranded DNA and 5′-overhangs, because this 5′-exonuclease is not dependent upon DNA-PKcs. Here, we show that the 5′-exonuclease and the endonuclease activities co-purify. Second, we show that a point mutant of ARTEMIS at a putative active site residue (H115A) markedly reduces both the endonuclease activity and the 5′-exonuclease activity. Third, divalent cation effects on the 5′-exonuclease and the endonuclease parallel one another. Fourth, both the endonuclease activity and 5′-exonuclease activity of ARTEMIS can be blocked in parallel by small molecule inhibitors, which do not block unrelated nucleases. We conclude that the 5′-exonuclease is intrinsic to ARTEMIS, making it relevant to the role of ARTEMIS in nonhomologous DNA end joining.

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.

Unifying the DNA end-processing roles of the artemis nuclease: Ku-dependent artemis resection at blunt DNA ends.

Background: Artemis is a nuclease that is necessary for hairpin opening in V(D)J recombination. Results: Artemis action on blunt DNA ends is dependent on DNA sequence (breathing) and Ku. Conclusion: Breathing of blunt DNA ends into a transient ss/dsDNA boundary is needed for Artemis action. Significance: Unification of Artemis nuclease action explains the features of NHEJ and V(D)J recombination. Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is essential in vertebrates because, during V(D)J recombination, the RAG complex generates hairpins when it creates the double strand breaks at V, D, and J segments, and Artemis is required to open the hairpins so that they can be joined. Artemis is a diverse endo- and exonuclease, and creating a unified model for its wide range of nuclease properties has been challenging. Here we show that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone because of a requirement for DNA end breathing (and confirmed using fixed pseudo-Y structures). All DNA ends are cut when both the DNA-dependent protein kinase catalytic subunit and Ku accompany Artemis but not when Ku is omitted. These are the first biochemical data demonstrating a Ku dependence of Artemis action on DNA ends of any configuration. The action of Artemis at blunt DNA ends is slower than at overhangs, consistent with a requirement for a slow DNA end breathing step preceding the cut. The AT sequence dependence, the order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis action at blunt ends can be reconciled with the other nucleolytic properties of both Artemis and Artemis·DNA-PKcs in a model incorporating DNA end breathing of blunt ends to form transient single to double strand boundaries that have structural similarities to hairpins and fixed 5′ and 3′ overhangs.

Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex

Abstract Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets.

Bridging of double-stranded breaks by the nonhomologous end-joining ligation complex is modulated by DNA end chemistry

Abstract The nonhomologous end-joining (NHEJ) pathway is the primary repair pathway for DNA double strand breaks (DSBs) in humans. Repair is mediated by a core complex of NHEJ factors that includes a ligase (DNA Ligase IV; L4) that relies on juxtaposition of 3΄ hydroxyl and 5΄ phosphate termini of the strand breaks for catalysis. However, chromosome breaks arising from biological sources often have different end chemistries, and how these different end chemistries impact the way in which the core complex directs the necessary transitions from end pairing to ligation is not known. Here, using single-molecule FRET (smFRET), we show that prior to ligation, differences in end chemistry strongly modulate the bridging of broken ends by the NHEJ core complex. In particular, the 5΄ phosphate group is a recognition element for L4 and is critical for the ability of NHEJ factors to promote stable pairing of ends. Moreover, other chemical incompatibilities, including products of aborted ligation, are sufficient to disrupt end pairing. Based on these observations, we propose a mechanism for iterative repair of DSBs by NHEJ.

DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining

Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends.

Effects of DNA end configuration on XRCC4-DNA ligase IV and its stimulation of Artemis activity.

In humans, nonhomologous DNA end-joining (NHEJ) is the major pathway by which DNA double-strand breaks are repaired. Recognition of each broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative binding of nucleases, DNA polymerases, and the XRCC4-DNA ligase IV (X4-LIV) complex in an order influenced by the configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining efficiency by functioning in a complex with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) that carries out endonucleolytic cleavage of 5′ and 3′ overhangs. Previously, we observed that X4-LIV alone can stimulate Artemis activity on 3′ overhangs, but this DNA-PKcs-independent endonuclease activity of Artemis awaited confirmation. Here, using in vitro nuclease and ligation assays, we find that stimulation of Artemis nuclease activity by X4-LIV and the efficiency of blunt-end ligation are determined by structural configurations at the DNA end. Specifically, X4-LIV stimulated Artemis to cut near the end of 3′ overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not able to stimulate Artemis activity at hairpins or at 5′ overhangs. We also found that X4-LIV and DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3′ overhangs, favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to DNA ends. These data suggest specific functional and positional relationships among these components that explain genetic and molecular features of NHEJ and V(D)J recombination within cells.