SLIDE 1
Mechanisms of DNA Repair by Photolyase and Excision Nuclease
Aziz Sancar Department of Biochemistry and Biophysics University of North Carolina School of Medicine Chapel Hill, North Carolina
1
2
Outline
Thymine Dimer (T<>T)
UV
Mechanisms of DNA Repair by Photolyase and Excision Nuclease Nobel - - PowerPoint PPT Presentation
Mechanisms of DNA Repair by Photolyase and Excision Nuclease Nobel - - PowerPoint PPT Presentation
Mechanisms of DNA Repair by Photolyase and Excision Nuclease Nobel Lecture in Chemistry Stockholms Universitet December 8, 2015 Aziz Sancar Department of Biochemistry and Biophysics University of North Carolina School of Medicine Chapel
SLIDE 2
SLIDE 3
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Outline
Thymine Dimer (T<>T)
SLIDE 4
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Photoreactivation (DNA Repair)
Sancar A and Rupert CS (1978) Gene 4:295-308
Light Dark
Rupert and Sancar, UT Dallas, 2009
T-T
PL+Blue
UV
T<>T
Thymine Dimer
UV Dose (J/m2) Surviving Fraction
SLIDE 5
Cloning and Purification of Photolyase
Electron micrograph of the plasmid containing Phr
Sancar A (1977) PhD Dissertation, UT Dallas
Purified photolyase protein has bright blue color
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Sancar A, et al (1984) JBC 259:6028-32 Sancar A and Sancar GB (1984) JMB 172:223-7
SLIDE 6
Photolyase Contains Two Cofactors
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Tan C, et al (2014) J Phys Chem A 118:10522-30
FAD (catalyst) Folate (solar panel)
SLIDE 7
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Structure of Photolyase
Park HW, et al (1995) Science 268:1866-1872
FADH- Folate
SLIDE 8
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Liu Z, et al (2011) PNAS 108:14831-36 Tan C, et al (2014) J Phys Chem A 118:10522-30
Reaction Mechanism of Photolyase
SLIDE 9
Ultrafast Kinetics of Photolyase
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Liu Z, et al (2011) PNAS 108:14831-36 Tan C, et al (2014) J Phys Chem A 118:10522-30
SLIDE 10
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Outline
Thymine Dimer (T<>T)
SLIDE 11
Model for UV Repair Circa 1982
11
+UV
5’ Endonuclease
(E. coli UvrA,B,C) (Human XPA-XPG)
Resynthesis 5’-3’ Exonuclease
- Thymine dimers are
removed from the genome in both E. coli and humans.
- Excised thymine dimers
were reported to exist in
- ligonucleotides 4-6 nt in
length.
- Excision is genetically
controlled by Uvr genes in
- E. coli and XP genes in
humans.
- Following excision, the
repair gap is filled in and ligated.
- Excised dimers remain
within the cell.
4-6-mer
SLIDE 12
Identification of the E. coli Excision Repair Proteins by the Maxicell Method
Sancar A et al (1981) JMB 148:63-76
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Sancar A et al (1981) PNAS 78:5450-54 Sancar A et al (1981) JMB 148:45-62
UvrA UvrB UvrC
Sancar A et al (1979) J Bacteriol 137:692-93
SLIDE 13
Purification of E. coli Excision Repair Proteins
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Sancar A and Rupp WD (1983) Cell 33:249-60
UvrA UvrB UvrC
SLIDE 14
Dual Incisions in E. coli Excision Repair
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Sancar A (1994) Science 266:1954-56
3’ incision 5’ incision
12-mer
T<>T
Sancar A and Rupp WD (1983) Cell 33:249-60
Excision Resynthesis
SLIDE 15
Mechanism of Excision Repair in E. coli
Lin JJ & Sancar A (1992) Mol Microbiol 6:2219-24
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SLIDE 16
Mechanism of Transcription Coupled Repair
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Selby CP & Sancar A (1993) Science 260:53-58
SLIDE 17
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Outline
Thymine Dimer (T<>T)
SLIDE 18
Model for UV Repair Circa 1982
18
+UV
5’ Endonuclease
(E. coli UvrA,B,C) (Human XPA-XPG)
Resynthesis 5’-3’ Exonuclease
- Thymine dimers are
removed from the genome in both E. coli and humans.
- Excised thymine dimers
were reported to exist in
- ligonucleotides 4-6 nt in
length.
- Excision is genetically
controlled by Uvr genes in
- E. coli and XP genes in
humans.
- Following excision, the
repair gap is filled in and ligated.
- Excised dimers remain
within the cell.
4-6-mer
SLIDE 19
Xeroderma Pigmentosum
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Halpern J, et al (2008) Cases J 1:254
Patients lacking excision repair XP proteins (XPA-XPG) have 5,000 higher incidence of skin cancer
SLIDE 20
Human Excision Repair Factors
Mu D, Hsu DS, Sancar A (1996) J Biol Chem 271:8285-94
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Mu D, et al (1995) J Biol Chem 270:2415-18
SLIDE 21
Dual Incisions in human Excision Repair
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Sancar A (1994) Science 266:1954-56
30-mer
3’ incision 5’ incision T<>T
Huang JC, et al (1992) PNAS 89:3664-68
Excision Resynthesis
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Reardon JT & Sancar A (2004) Cell Cycle 3:141-4
Mechanism of Excision Repair in Humans
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Mapping the Excised Oligomer in Humans
Hu J, Adar S, et al (2015) Genes Dev 29:948-60
Lysate from UV-irradiated cells Ds DNA library with barcodes Next Generation Sequencing (NGS)
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Excision Repair Map of the Human Genome
Hu J, Adar S, et al (2015) Genes Dev 29:948-60
SLIDE 25
Excision Repair of p53 at Single Nucleotide Resolution
Chr17:
RNA Repair
+ +
- 20Mb
Megabase
Cancer-linked Mutation
Nucleotide
5’-AGCTGTTCCGTCCCAGTAGATTACCA
7,577,150 7,577,151
10Kb
RNA Repair
+ +
- Repair at the p53 gene:
WRAP53 p53
Kilobase
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Hu J, Adar S, et al (2015) Genes Dev 29:948-60
SLIDE 26
Excision Repair
- Nucleotide excision repair is initiated
by dual incisions in both E. coli and humans.
- Excision is genetically controlled by the
evolutionarily unrelated Uvr genes in
- E. coli and XP genes in humans.
- Dual incisions remove an oligomer of
~12 nucleotides in E. coli and ~30 nucleotides in humans.
- Following excision, the repair gap is
filled in and ligated.
- By capturing the excised oligomers, we
have generated an excision repair map
- f the whole human genome.
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T<>T
- E. coli
Human
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Outline
Thymine Dimer (T<>T)
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- Humans do not have photolyase
Li YF, et al (1993) PNAS 90:4389-93
- Humans have a photolyase homolog
Adams MD, et al (1995) Nature 377:3-174
Cryptochrome
Photolyase Homology Domain 1 472
- E. coli Photolyase
Human Cryptochrome 1
1 586 Photolyase Homology Domain
Human Cryptochrome 2
1 593 Photolyase Homology Domain
hCRY 1 Photolyase hCRY 2
Cryptochrome Photolyase
Brautigam CA, et al (2004) PNAS 101:12142-47
- Humans have 2 photolyase paralogs
Hsu DS, et al (1996) Biochemistry 35:13871-77
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www.NASA.gov
Jetlag, Cryptochrome, and the Circadian Clock
Determined that human CRYs are not repair proteins Traveled to Turkey to visit family and
- n my return flight read the AA
Inflight Magazine article by William Schwartz, “Internal Timekeeping” about jetlag and the circadian clock
Spring 1996 May - June 1996
Wrote the human CRY paper claiming CRYs are circadian proteins Discovered genetic evidence that human CRYs are clock proteins
June - August 1996 May - November 1998
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Clock and Circadian Clock
- Clock is a Time Keeping Object/System
- Mechanic
- Electronic
- Molecular (Circadian Clock)
- Circadian Clock is an innate timekeeping
molecular mechanism that maintains daily rhythmicity in biochemical, physiological and behavioral functions independent of external input.
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Cryptochrome is Essential for the Circadian Clock WT Cry1-/- Cry2-/- Cry1-/- Cry2-/-
Thresher RJ, et al (1998) Science 282:1490-94 Vitaterna MH, et al (1999) PNAS 96:12114-19
48 Hours 1 28 Days 1 28 Days 1 28 Days 1 28 Days DD LD DD LD DD LD DD LD
SLIDE 32
1) CRYPTOCHROME (Flavoprotein) 2) PERIOD (PAS domain) 3) CLOCK (bHLH-PAS) 4) BMAL1 (bHLH-PAS)
Mammalian Clock Genes/Proteins (1996-2000)
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Circadian Control Mechanism
Sancar A, et al (2010) FEBS Lett 584:2618-25
Clock Controlled Genes
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Circadian Control of Excision Repair
Kang T, et al (2010) PNAS 107:4890-95 Gaddameedhi S, et al (2011) PNAS 108:18790-95
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Photolyase
Summary
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WT Cry1-/- Cry2-/- Cry1-/- Cry2-/-
Cryptochrome
- E. coli
Human
Nucleotide Excision Repair
3’ 5’ 3’ 5’
T<>T
SLIDE 36
Acknowledgments
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Sancar Lab Members
Adar, Sheera Ahn, Kyujeong Akan, Zafer Annayev, Yunus Araujo, Francisco Arat, Nezahat Arnette, Robin Asimgil, Hande Bereketoglu, Sidar Berrocal, Gloria Bessho, Tadayoshi Bondo, Eddie Bouyer, James Branum, Mark Cakit, Ceylan Cantürk, Fazile Capp, Christopher Carlton, Wendi Chiou, Yi-Ying Choi, Jun-Hyuk Croteau, Deborah Dawut, Lale Denaro, Tracy DeRocco, Vanessa Ensch-Simon, Ingrid Erkmen, Gulnihal Kulaksiz Gaddameedhi, Shobhan Gauger, Michele Han, Chih-Chiang (Eric) Hara, Ryujiro Hassan, Bachar Heenan, Erin Hsu, Shiao-Wen (David) Hu, Jinchuan Huang, Juch-Chin (JC) Husain, Intisar Hutsell, Stephanie Jiang, Gouchun Kang, Tae-Hong Karaman, Muhammet Kavakli, Ibrahim (Halil) Kawara, Hiroaki Kazantsev, Aleksey Kemp, Michael Kim, Sang-Tae Lee, Jin-Hyup Levy, Michael Li, Wentao Li, Ywan-Feng Lin, Jing-Jer Lindsey-Boltz, Laura Malhotra, Khushbeer Matsunaga, Tsukasa McDowell-Buchanan, Carla Meganck, Rita Miyamoto, Yasuhide Mo, Jinyao Morrison, Lydia Mu, David Myles, Gary Nichols, Anne Ögrünç, Müge Orren, David Özer, Zahide Özgür, Sezgin Ozkan-Dagliyan, Irem Öztürk, Nuri Park, Chi-Hyun Partch, Carrie Payne, Gillian Payne, Nicola Petit, Claude Phillips, A. Meleah Rastogi, Promila Reardon, Joyce Sar, Funda Selby, Christopher Sercin, Ozdemirhan Shields, Katie Sibghat-Ullah Smith, Frances Song, Sang-Hun Svoboda, Daniel Thomas, David Thompson, Carol Thresher, Randy Ünsal-Kaçmaz, Keziban Vagas, Elif Van Houten, Ben Wakasugi, Mitsuo Worthington, Erin (Nikki) Yang, Yanyan Ye, Rui Yilmaz, Seçil Zhao, Xiaodong (Jerry) Zhao, Shaying
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RUPERT, CLAUD S. Aksoy, Muzaffer Rupp, W. Dean Howard-Flanders, Paul
Mentors Funding Collaborators
Bambara, Robert Chaney, Stephen Cordeiro-Stone, Marila Deisenhofer, Johann Griffith, Jack Hearst, John Heelis, Paul Hurwitz, Jerard Jorns, Marilyn Kaufmann, William Kunkel, Thomas Lieb, Jason Linn, Stuart Lippard, Stephen Modrich, Paul Rajagopalan, K.V. Reinberg, Danny Sancar, Gwendolyn Smithies, Oliver Takahashi, Joseph Taylor, John-Stephen Thompson, Larry Van Gelder, Russel Wold, Marc Zhong, Dongping
Contributors
Photolyase
Eker, Andries Sancar, Gwendolyn Todo, Takeshi Yasui, Akira
Excision Repair
Cleaver, James Egly, Jean-Marc Friedberg, Errol Goosen, Nora Grossman, Larry Hanaoka, Fumio Hanawalt, Philip Hoeijmakers, Jan
Circadian Clock
Provencio, Ignacio Reppert, Steven Rosbash, Michael Sassone-Corsi, Paolo Schibler, Ueli Takahashi, Joseph van der Horst, Gijsbertus Young, Michael Kisker, Caroline Prakash, Louise Prakash, Satya Tanaka, Kiyoji Thompson, Larry Van Houten, Ben Witkin, Evelyn Wood, Richard