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ABSTRACT: DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation.
International journal of radiation oncology, biology, physics 02/2013; · 4.59 Impact Factor
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ABSTRACT: DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions.
Biochemistry and Cell Biology 02/2013; 91(1):31-41. · 2.67 Impact Factor
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ABSTRACT: Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 in order to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically-disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to forbidden residues K,R,H and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to tune the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the non-homologous end-joining (NHEJ) DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically-disordered proteins and their binding partners.
Molecular & Cellular Proteomics 11/2012; · 7.40 Impact Factor
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Cell cycle (Georgetown, Tex.) 10/2012; 11(22). · 5.36 Impact Factor
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Chris T Williamson,
Eiji Kubota,
Jeffrey D Hamill,
Alexander Klimowicz,
Ruiqiong Ye,
Huong Muzik,
Michelle Dean,
LiRen Tu,
David Gilley,
Anthony M Magliocco,
Bruce C McKay,
D Gwyn Bebb, Susan P Lees-Miller
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ABSTRACT: Poly-ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia-telangiectasia mutated (ATM)-deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM-deficient MCL cells, olaparib induced DNA-PK-dependent phosphorylation and stabilization of p53 as well as expression of p53-responsive cell cycle checkpoint regulators, and inhibition of DNA-PK reduced the toxicity of olaparib in ATM-deficient MCL cells. Thus, both DNA-PK and p53 regulate the response of ATM-deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53-deficient malignancies.
EMBO Molecular Medicine 03/2012; 4(6):515-27. · 10.33 Impact Factor
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ABSTRACT: XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together--stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.
Nucleic Acids Research 02/2012; 40(4):1684-94. · 8.03 Impact Factor
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ABSTRACT: The DNA-dependent protein kinase (DNA-PK) was identified as an activity and as its three component polypeptides 25 and 15 years ago, respectively. It has been exhaustively characterized as being absolutely dependent on free double stranded DNA ends (to which it is directed by its regulatory subunit, Ku) for its activation as a robust nuclear serine/threonine protein kinase. Here, we report the unexpected finding of robust DNA-PKcs activation by N-terminal constraint, independent of either DNA or its regulatory subunit Ku. These data suggest that an N-terminal conformational change (likely induced by DNA binding) induces enzymatic activation.
Nucleic Acids Research 12/2011; 40(7):2964-73. · 8.03 Impact Factor
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Michal Hammel,
Martial Rey,
Yaping Yu,
Rajam S. Mani,
Scott Classen,
Mona Liu,
Michael E. Pique,
Shujuan Fang,
Brandi L. Mahaney,
Michael Weinfeld,
David C. Schriemer, Susan P. Lees-Miller,
John A. Tainer
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ABSTRACT: The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double
strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic.
Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define
the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain
interfaces forming parallel super-helical filaments. XLF Leu-115 (“Leu-lock”) inserts into a hydrophobic pocket formed by
XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive
specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively
charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact
to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation
of DNA ligation.
Journal of Biological Chemistry 09/2011; 286(37):32638-32650. · 4.77 Impact Factor
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Angela E Zolner,
Ismail Abdou,
Ruiqiong Ye,
Rajam S Mani,
Mesfin Fanta,
Yaping Yu,
Pauline Douglas,
Nasser Tahbaz,
Shujuan Fang,
Tracey Dobbs,
Chen Wang,
Nick Morrice,
Michael J Hendzel,
Michael Weinfeld, Susan P Lees-Miller
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ABSTRACT: Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5'-DNA kinase/3'-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.
Nucleic Acids Research 08/2011; 39(21):9224-37. · 8.03 Impact Factor
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Michal Hammel,
Martial Rey,
Yaping Yu,
Rajam S Mani,
Scott Classen,
Mona Liu,
Michael E Pique,
Shujuan Fang,
Brandi L Mahaney,
Michael Weinfeld,
David C Schriemer, Susan P Lees-Miller,
John A Tainer
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ABSTRACT: The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 ("Leu-lock") inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.
Journal of Biological Chemistry 07/2011; 286(37):32638-50. · 4.77 Impact Factor
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ABSTRACT: How a cell chooses between nonhomologous end joining (NHEJ) and homologous recombination (HR) to repair a double-strand break (DSB) is a central and largely unanswered question. Although there is evidence of competition between HR and NHEJ, because of the DNA-dependent protein kinase (DNA-PK)'s cellular abundance, it seems that there must be more to the repair pathway choice than direct competition. Both a mutational approach and chemical inhibition were utilized to address how DNA-PK affects HR. We find that DNA-PK's ability to repress HR is both titratable and entirely dependent on its enzymatic activity. Still, although requisite, robust enzymatic activity is not sufficient to inhibit HR. Emerging data (including the data presented here) document the functional complexities of DNA-PK's extensive phosphorylations that likely occur on more than 40 sites. Even more, we show here that certain phosphorylations of the DNA-PK large catalytic subunit (DNA-PKcs) clearly promote HR while inhibiting NHEJ, and we conclude that the phosphorylation status of DNA-PK impacts how a cell chooses to repair a DSB.
Molecular and cellular biology 02/2011; 31(8):1719-33. · 6.06 Impact Factor
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Rajam S Mani,
Yaping Yu,
Shujuan Fang,
Meiling Lu,
Mesfin Fanta,
Angela E Zolner,
Nasser Tahbaz,
Dale A Ramsden,
David W Litchfield, Susan P Lees-Miller,
Michael Weinfeld
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ABSTRACT: XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.
Journal of Biological Chemistry 11/2010; 285(48):37619-29. · 4.77 Impact Factor
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Rajam S. Mani,
Yaping Yu,
Shujuan Fang,
Meiling Lu,
Mesfin Fanta,
Angela E. Zolner,
Nasser Tahbaz,
Dale A. Ramsden,
David W. Litchfield, Susan P. Lees-Miller,
Michael Weinfeld
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ABSTRACT: XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold
protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes
a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase
(PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within
the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited
PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent
of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different
effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.
Journal of Biological Chemistry 11/2010; 285(48):37619-37629. · 4.77 Impact Factor
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ABSTRACT: Prolonged exposure to estrogen increases breast cancer risk. Estrogen is known to induce chromosomal aberrations, yet the mechanisms by which estrogen promotes genomic instability are not fully understood. Here, we show that exposure of MCF-7 cells to 17β-estradiol (E2) induces DNA double-strand breaks (DSBs), as determined by the formation of γH2AX foci. Foci formation was dependent upon estrogen receptor-α (ERα) and the catalytic activity of the type II topoisomerase, topoisomerase IIβ (topoIIβ). Moreover, we show by chromatin immunoprecipitation that topoIIβ-dependent E2-induced γH2AX localizes to the promoter of the estrogen-inducible gene, trefoil factor 1. E2-induced foci were associated with cyclin A expression and inhibited by pre-incubation with the DNA polymerase inhibitor aphidicolin suggesting that E2-induced DSBs are mediated by progression through S phase. Furthermore, E2-induced γH2AX foci colocalized with Rad51, suggesting that E2-induced DSBs are repaired by homologous recombination. We propose that DNA DSBs formed by the strand-cleaving activity of the topoIIβ-DNA cleavage complex at estrogen-inducible genes can present a barrier to DNA replication, leading to persistent DNA DSBs in ERα-positive breast cancer cells.
Carcinogenesis 11/2010; 32(3):279-85. · 5.70 Impact Factor
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ABSTRACT: DNA ligase IV (LigIV) is critical for nonhomologous end joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in human cells, and LigIV activity is regulated by XRCC4 and XLF (XRCC4-like factor) interactions. Here, we employ small angle X-ray scattering (SAXS) data to characterize three-dimensional arrangements in solution for full-length XRCC4, XRCC4 in complex with LigIV tandem BRCT domains and XLF, plus the XRCC4·XLF·BRCT2 complex. XRCC4 forms tetramers mediated through a head-to-head interface, and the XRCC4 C-terminal coiled-coil region folds back on itself to support this interaction. The interaction between XLF and XRCC4 is also mediated via head-to-head interactions. In the XLF·XRCC4·BRCT complex, alternating repeating units of XLF and XRCC4·BRCT place the BRCT domain on one side of the filament. Collective results identify XRCC4 and XLF filaments suitable to align DNA molecules and function to facilitate LigIV end joining required for DSB repair in vivo.
Structure 11/2010; 18(11):1431-42. · 6.35 Impact Factor
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ABSTRACT: The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku heterodimer together form the biologically critical DNA-PK complex that plays key roles in the repair of ionizing radiation-induced DNA double-strand breaks through the non-homologous end-joining (NHEJ) pathway. Despite elegant and informative electron microscopy studies, the mechanism by which DNA-PK co-ordinates the initiation of NHEJ has been enigmatic due to limited structural information. Here, we discuss how the recently described small angle X-ray scattering structures of full-length Ku heterodimer and DNA-PKcs in solution, combined with a breakthrough DNA-PKcs crystal structure, provide significant insights into the early stages of NHEJ. Dynamic structural changes associated with a functionally important cluster of autophosphorylation sites play a significant role in regulating the dissociation of DNA-PKcs from Ku and DNA. These new structural insights have implications for understanding the formation and control of the DNA-PK synaptic complex, DNA-PKcs activation and initiation of NHEJ. More generally, they provide prototypic information for the phosphatidylinositol-3 kinase-like (PIKK) family of serine/threonine protein kinases that includes Ataxia Telangiectasia-Mutated (ATM) and ATM-, Rad3-related (ATR) as well as DNA-PKcs.
DNA repair 10/2010; 9(12):1307-14. · 4.20 Impact Factor
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ABSTRACT: Repair and integrity of DNA ends at breaks, replication forks and telomeres are essential for life; yet, paradoxically, these responses are, in many cases, controlled by a single protein complex, Mre11-Rad50-Nbs1 (MRN). The MRN complex consists of dimers of each subunit and this heterohexamer controls key sensing, signaling, regulation, and effector responses to DNA double-strand breaks including ATM activation, homologous recombinational repair, microhomology-mediated end joining and, in some organisms, non-homologous end joining. We propose that this is possible because each MRN subunit can exist in three or more distinct states; thus, the trimer of MRN dimers can exist in a stunning 6(3) or 216 states, a number that can be expanded further when post-translational modifications are taken into account. MRN can therefore be considered as a molecular computer that effectively assesses optimal responses and pathway choice based upon its states as set by cell status and the nature of the DNA damage. This extreme multi-state concept demands a paradigm shift from striving to understand DNA damage responses in separate terms of signaling, checkpoint, and effector proteins: we must now endeavor to characterize conformational and assembly states of MRN and other DNA repair machines that couple, coordinate, and control biological outcomes. Addressing the emerging challenge of gaining a detailed molecular understanding of MRN and other multi-state dynamic DNA repair machines promises to provide opportunities to develop master keys for controlling cell biology with probable impacts on therapeutic interventions.
DNA repair 10/2010; 9(12):1299-306. · 4.20 Impact Factor
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Aging 05/2010; 2(5):257-8. · 5.13 Impact Factor
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Cell cycle (Georgetown, Tex.) 04/2010; 9(7):1221-2. · 5.36 Impact Factor
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ABSTRACT: Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl(2) concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding.
Journal of Biological Chemistry 03/2010; 285(23):17778-88. · 4.77 Impact Factor
