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ABSTRACT: How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.
Journal of Biological Chemistry 02/2012; 287(15):12036-49. · 4.77 Impact Factor
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ABSTRACT: This chapter describes a technique in which indirect immunofluorescence is applied to visualize the process of nucleotide excision repair (NER) at the site of locally induced damage in DNA. UV-irradiation of cells through an isopore polycarbonate membrane filter generates cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP) on a subnuclear area, which corresponds to the size of a pore on the membrane. Specific antibodies to CPD and 6-4PP define the damaged spot. The NER components co-localize at the damaged-DNA subnuclear spot, where the proteins are stained with the appropriate fluorescent antibodies. This relatively simple and affordable method facilitates the examination of the sequential assembly of NER proteins in the chromatin-embedded DNA photoproducts. The method also enhances the identification of repair auxiliary proteins and complexes, such as ubiquitin E3 ligases, involved in the initiation of NER on non-transcribed DNA.
Methods in molecular biology (Clifton, N.J.) 01/2011; 682:149-61.
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Academic medicine: journal of the Association of American Medical Colleges 09/2010; 85(9 Suppl):S501-4. · 2.34 Impact Factor
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Michael Boninger,
Philip Troen,
Emily Green,
Jeffrey Borkan,
Cynthia Lance-Jones,
Allen Humphrey,
Philip Gruppuso,
Peter Kant,
James McGee,
Michael Willochell,
Nina Schor,
Steven L Kanter, Arthur S Levine
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ABSTRACT: An increasing number of medical schools have implemented or are considering implementing scholarly activity programs as part of their undergraduate medical curricula. The goal of these programs is to foster students' analytical skills, enhance their self-directed learning and their oral and written communication skills, and ultimately to train better physicians. In this article, the authors describe the approach to implementing scholarly activities at a school that requires this activity and at a school where it is elective. Both programs have dealt with significant challenges including orienting students to a complex activity that is fundamentally different than traditional medical school courses and clerkships, helping both students and their mentors understand how to "stay on track" and complete work, especially during the third and fourth years, and educating students and mentors about the responsible conduct of research, especially involving human participants. Both schools have found the implementation process to be evolutionary, requiring experience before faculty could significantly improve processes. A required scholarly activity has highlighted the need for information technology (IT) support, including Web-based document storage and student updates, as well as automatic e-mails alerting supervisory individuals to student activity. Directors of the elective program have found difficulty with both ensuring uniform outcomes across different areas of study and leadership changes in a process that has been largely student-driven. Both programs have found that teamwork, regular meetings, and close communication have helped with implementation. Schools considering the establishment of a scholarly activity should consider these factors when designing programs.
Academic medicine: journal of the Association of American Medical Colleges 03/2010; 85(3):429-37. · 2.34 Impact Factor
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ABSTRACT: In the synergistic evolution of their research, educational, and clinical programs, the University of Pittsburgh (Pitt) School of Medicine (SOM) and the University of Pittsburgh Medical Center (UPMC) have followed one core principle: What is good for one is good for both. The collaboration is underpinned by UPMC's commitment to its community mission, including support for the academic and research objectives of the SOM. UPMC's conceptual origin was fostered by its experience with Western Psychiatric Institute and Clinic in the 1970s. Over time, UPMC acquired other hospitals through merger and negotiation and, by 2008, had grown into a $7 billion global health enterprise. From the outset, the senior leaders of both UPMC and Pitt committed to collaborative decision making on all key issues. Under this coordinated decision-making model, UPMC oversees all clinical activity, including that from a consolidated physicians' practice plan. Pitt remains the guardian of all academic priorities, particularly faculty-based research. UPMC's steady financial success underpins the model. A series of interrelated agreements formally defines the relationship between Pitt and UPMC, including shared board seats and UPMC's committed ongoing financial support of the SOM. In addition, the two institutions have jointly made research growth a priority. The payoff from this dynamic has been a steadily growing Pitt research portfolio; enhanced growth, visibility, and stature for UPMC, the SOM, and Pitt as a whole; and the sustained success of UPMC's clinical enterprise, which now has an international scope. Given the current stagnation in the National Institutes of Health budget, the Pitt-UPMC experience may be instructive to other academic health centers.
Academic medicine: journal of the Association of American Medical Colleges 10/2008; 83(9):816-26. · 2.34 Impact Factor
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ABSTRACT: By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)-based ubiquitin ligase, DDB1-CUL4A(DDB2). We previously showed that DDB1-CUL4A(DDB2) ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A(DDB2) and DDB1-CUL4B(DDB2). To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B(DDB2) E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B(DDB2) is more efficient than DDB1-CUL4A(DDB2) in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B(DDB2) E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER.
Cancer Research 08/2008; 68(13):5014-22. · 7.86 Impact Factor
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ABSTRACT: Abraham Flexner persuaded the medical establishment of his time that teaching the sciences, from basic to clinical, should be a critical component of the medical student curriculum, thus giving rise to the "preclinical curriculum." However, students' retention of basic science material after the preclinical years is generally poor. The authors believe that revisiting the basic sciences in the fourth year can enhance understanding of clinical medicine and further students' understanding of how the two fields integrate. With this in mind, a return to the basic sciences during the fourth year of medical school may be highly beneficial. The purpose of this article is to (1) discuss efforts to integrate basic science into the clinical years of medical student education throughout the United States and Canada, and (2) describe the highly developed fourth-year basic science integration program at the University of Pittsburgh School of Medicine. In their critical review of medical school curricula of 126 U.S. and 17 Canadian medical schools, the authors found that only 19% of U.S. medical schools and 24% of Canadian medical schools require basic science courses or experiences during the clinical years, a minor increase compared with 1985. Curricular methods ranged from simple lectures to integrated case studies with hands-on laboratory experience. The authors hope to advance the national discussion about the need to more fully integrate basic science teaching throughout all four years of the medical student curriculum by placing a curricular innovation in the context of similar efforts by other U.S. and Canadian medical schools.
Academic medicine: journal of the Association of American Medical Colleges 08/2008; 83(7):662-9. · 2.34 Impact Factor
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ABSTRACT: Today's medical student curriculum is a lock-step experience that provides a broad survey of medicine with little opportunity to pursue fully integrated, in-depth learning. To teach students about the human dimensions of health care, many schools simply have added courses that survey general areas such as ethics, values, and patient-doctor relationships. However, a superficial, broad-brush approach does not offer students sufficient opportunity to engage with these topics in substantive and meaningful ways. The authors propose that a theme-based, individualized, in-depth learning experience (in which students pursue a focused project comprehensively and in detail)--one that is an integral part of the curriculum--helps students learn to blend values and ethics with medicine in a way that cannot occur during rapid-paced topical survey courses. Furthermore, it is in the depths of a learning experience that one comes face to face with the realities of uncertainty: the realization that unanswerable questions outnumber answerable ones; the awareness of the difficulty in accumulating sufficient evidence to answer a question that is, in fact, answerable; the recognition that many patients' problems transcend available evidence and must be addressed by the art of medicine; the realization that a patient can have a condition that one cannot diagnose and that may even get better for reasons that one cannot understand. The authors describe three initiatives at the University of Pittsburgh School of Medicine, two of which have been offered for more than 10 years, that illustrate the value of in-depth learning experiences. These in-depth experiences blend situated learning, reflective exercises, faculty mentoring, critical reading of literature, and constructive feedback in a prescribed but individualized curriculum.
Academic Medicine 05/2007; 82(4):405-9. · 3.52 Impact Factor
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ABSTRACT: Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4A(DDB2). The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4A(DDB2) complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4A(DDB2) ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.
Proceedings of the National Academy of Sciences 03/2006; 103(8):2588-93. · 9.68 Impact Factor
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ABSTRACT: Many U.S. medical schools offer students the opportunity to undertake laboratory or clinical research or another form of scholarly project over the summer months, yet few require this as a prerequisite for graduation, and even fewer provide comprehensive didactic material in preparation for the performance of such a project as an integrated component of their curricula. The authors describe the Scholarly Project Initiative of the University of Pittsburgh School of Medicine, a novel, longitudinal, and required program. The program will aim to provide all students with structured preparatory coursework, foster critical analytical and communication skills, and introduce the breadth and depth of the research and scholarly enterprise engendered by modern academic medicine in the contexts of both the classroom and an individual, mentored experience. The initiative has two goals: encouraging an interest in academic medicine in an era marked by the continuing decline in the number of physician-investigators, and fostering the development of physicians who have confidence in their abilities to practice medicine with creativity, original and analytical thought, and relentless attention to the scientific method. Planning for the Scholarly Project Initiative began officially at the University of Pittsburgh School of Medicine's Curriculum Colloquium in May 2003. The initiative was implemented with the first-year class of July 2004 as part of the new "Scientific Reasoning and Medicine" block of the School of Medicine's curriculum. The block as a whole includes traditional lectures, small-group laboratory and problem-based sessions, and mentored independent study components.
Academic Medicine 10/2005; 80(9):824-31. · 3.52 Impact Factor
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ABSTRACT: Xeroderma pigmentosum (XP) is a skin cancer-prone autosomal recessive disease characterized by inability to repair UV-induced DNA damage. The major form of XP is defective in nucleotide excision repair (NER) and comprises seven complementation groups (A-G). The genes defective in all groups have been identified unambiguously with the exception of group E. The cells of some XP-E patients are deficient in a protein complex (consisting of two subunits: p127/DDBI and p48/DDB2) which binds to UV-damaged DNA (UV-DDB) and is specifically involved in the removal of photoproducts from the non-transcribed regions of the genome. However, other XP-E patients have been reported not to lack UV-damaged DNA binding activity (DDB(+)). Here we describe several genetically unrelated XP-E patients, not previously analyzed in depth, each carrying two mutated alleles for DDB2, causing either a single amino acid change or a protein truncation or internal deletion. These defects result in a severe decrease of detectable p48 protein, abolish interaction with the p127 subunit, and produce a deficiency in UV-DDB binding activity (DDB(-)). The role of p48 in the repair defect of these patients was demonstrated in vivo and in vitro. Investigation of four DDB(+) cell strains from patients previously assigned to XP-E, allowed us to reclassify all of them into other groups and to show that they do not share the molecular and biochemical features typical for XP-E. Besides confirming that the true XP-E phenotype is DDB(-), resulting from defects in a single gene, DDB2, our results identify the functional domains of the corresponding p48 protein.
Human Molecular Genetics 08/2003; 12(13):1507-22. · 7.64 Impact Factor
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ABSTRACT: The UV-damaged DNA binding protein complex (UV-DDB) is implicated in global genomic nucleotide excision repair (NER) in mammalian cells. The complex consists of a heterodimer of p127 and p48. UV-DDB is defective in one complementation group (XP-E) of the heritable, skin cancer-prone disorder xeroderma pigmentosum. Upon UV irradiation of primate cells, UV-DDB associates tightly with chromatin, concomitant with the loss of extractable binding activity. We report here that an early event after UV, but not ionizing, radiation is the transient dose-dependent degradation of the small subunit, p48. Treatment of human cells with the proteasomal inhibitor NIP-L3VS blocks this UV-induced degradation of p48. In XP-E cell lines with impaired UV-DDB binding, p48 is resistant to degradation. UV-mediated degradation of p48 occurs independently of the expression of p53 and the cell's proficiency for NER, but recovery of p48 levels at later times (12 h and thereafter) is dependent upon the capacity of the cell to repair non-transcribed DNA. In addition, we find that the p127 subunit of UV-DDB binds in vivo to p300, a histone acetyltransferase. The data support a functional connection between UV-DDB binding activity, proteasomal degradation of p48 and chromatin remodeling during early steps of NER.
Nucleic Acids Research 07/2002; 30(11):2588-98. · 8.03 Impact Factor
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ABSTRACT: A cDNA which encodes á 127 kDa UV–damaged DNA–binding (UV-DDB) protein with high affinity for (6—4)pyrlmidine dimers [Abramic', M., Levine, A.S. & Protic', M., J. Biol. Chem. 266:22493—22500,1991] has been Isolated from a monkey cell cDNA library. The presence of this protein in complexes bound to UV-damaged DNA was confirmed by immunobiotting. The human cognate of the UV-DDB gene was localized to chromosome 11. UV-DDB mRNA was expressed in all human tissues examined, including cells from two patients with xeroderma pigmentosum (group E) that are deficient in UV-DDB activity, which suggests that the binding defect in these cells may reside in a dysfunctional UV-DDB protein. Database searches have revealed significant homology of the UV-DDB protein sequence with partial sequences of yet uncharacterized proteins from Dictyostellum discoldeum (44% Identity over 529 amino acids) and Oryza satlva (54% identity over 74 residues). According to our results, the UV-DDB polypeptide belongs to a highly conserved, structurally novel family of proteins that may be involved in the early steps of the UV response, e.g., DNA damage recognition.
