Aziz Sancar

Research: DNA repair and regulation of the circadian clock

Aziz Sancar

Sarah Graham Kenan Professor of Biochemistry and Biophysics
(PhD - University of Texas; MD - Istanbul University)

120 Mason Farm Rd, CB# 7260
3073 Genetic Medicine
Chapel Hill, NC 27599-7260
919-962-0115

Sancar Lab Website

HONORS AND AWARDS:

  • 1969    MD, Summa Cum Laude (1st in class of 625)
  • 1984    NSF Presidential Young Investigator Award
  • 1995    NIH MERIT Award
  • 2004    American Academy of Arts and Sciences
  • 2005    National Academy of Sciences, USA
  • 2006    Turkish Academy of Sciences
  • 2007    Turkish Koç Award
  • 2009    University of Texas at Dallas Distinguished Alumni Award
  • 2014    Distinguished Visiting Professor – Academia Sinica

RESEARCH INTERESTS:

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Fig. 1. The XR-seq method. (A) Schematic of nucleotide excision repair. (B) Excision patterns of photoproducts in wild-type, XP-C (deficient in global re-pair), and CS-B (deficient in TCR) cells. (C) Procedure for preparation of the dsDNA library for the Illumina HiSeq 2000 platform. (D) Distribution of the XR-seq signal, separated by strand, for CPD (top) and (6-4)PP (bot-tom) over a 1.5-Mb region of chromosome 3. (E) Strong association of TCR with RNA levels.

Our lab works on three interrelated subjects: (1) Mammalian DNA Excision Repair; (2) Mammalian DNA Damage Checkpoints; (3) Mammalian Circadian Clock.  Although all three systems mediate organismal response to external and internal signals so as to maintain homeostasis and adaptation to the environment by different mechanisms, they are at the organism level, coupled systems.

DNA Repair

DNA damage by exogenous physical and chemical agents is the most common cause of cancer.  Conversely, some of the most commonly used anticancer drugs kill malignant cells by damaging their DNA.  DNA Repair is the ensemble of molecular mechanisms that eliminate DNA damage from the genome, and it plays crucial roles in carcinogenesis and in cancer therapy.  Our lab works on Nucleotide Excision Repair which is the sole pathway for repairing cyclobutane pyrimidine dimers (CPDs) and cisplatin 1,2-d(GpG) adducts that cause cancer and cure cancer, respectively.  We discovered that these lesions are removed from DNA by dual incisions that generate 24-32 nucleotide-long oligomers (“nominal 30-mer”).  We identified and purified the 6 repair factors, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, that are essential for dual incisions.  Using these purified factors, we reconstituted human excision repair in vitro and defined the molecular mechanism of excision repair.  The in vitro work was complemented by in vivo studies which enabled us to generate a repair map of the entire genome.  We discovered that the nominal 30-mer is released in a tight complex with TFIIH, and this finding enabled us to isolate the nominal 30-mers from irradiated cells and subject them to deep sequencing (Fig. 1).  By using normal human fibroblasts and mutant cell lines we created repair maps for general repair and transcription-coupled repair of UV damage for the entire human genome through this method which we have named XR-seq (eXcision Repair-Sequencing).  Our future work will exploit XR-seq to uncover novel genomic regulators of excision repair of DNA damage by anti-cancer drugs with the ultimate goal of developing improved chemotherapy regimens.  In addition, we plan to investigate the effect of the excised nominal 30-mer on cellular physiology and the processing and ultimate fates of the excised oligonucleotides.

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Fig. 2. (A) Coupling Excision Repair and the ATR-mediated Checkpoint. When DNA is damaged by UV or a UV-mimetic agent, the core excision repair factors (RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1) excise a ∼30-nt oligomer containing the damage. The resulting gap is either filled in by polymerases or the gap is enlarged by EXO1. The enlarged ssDNA gap is coated with RPA, which recruits ATR-ATRIP, TopBP1, and substrates including p53. (B) Schematic of repair-checkpoint coupling in vitro. (C) Repair-checkpoint coupling Kinase reactions containing RPA as a substrate were incubated with ATR-ATRIP, TopBP1, and EXO1 as indicated. Kinase reactions containing unmodified (UM) or AAF-DNA from excision reactions with or without repair factors (RF) as indicated. Reactions were analyzed by immune-blotting for phospho-RPA2 (Ser33).

DNA Damage Checkpoints

DNA Damage Checkpoints are intracellular signaling pathways that delay or arrest cell cycle progression in response to DNA damage and thus aid in preventing mutations and cellular and organismic death.  In human cells, two mail DNA damage checkpoints pathways have been defined that are governed by the ATM and ATR protein kinases.  The ATR pathway is activated by UV or by replication block by base damage or by nucleotide depletion.  In line with our interest with cellular response to UV and UV-mimetic agents, we have been studying the ATR-mediated DNA damage checkpoint response using in vitro systems with purified proteins as well as cell-based assays.  Using purified proteins and model DNA substrates that mimic the initiating triggers for DNA damage checkpoint activation, we have reconstituted the basic ATR-dependent signaling reaction.  Importantly, we have succeeded in linking DNA excision repair to the ATR checkpoint pathway (Fig. 2): Using purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and damaged DNA, we coupled excision repair to the ATR-mediated checkpoint.  We found that checkpoint signaling as measured by phosphorylation of target proteins (RPA, p53) required the enlargement of the single-strand gap generated by the excision repair system by the 5’ to 3’ exonuclease activity of EXO1 enzyme in accordance with the in vivo data.  Future work will take advantage of our unique in vitro system to characterize novel regulators of ATR signaling and to screen and validate promising anti-cancer drugs that inhibit the ATR function. 

 

Cryptochrome and the Circadian Clock

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Fig. 3. Current Model for the Mammalian Circadian Clock. (A) Mammalian molecular clock model of the transcription-translation feedback loop (TTFL). (B) PER2 removes CRY1 from the CRY1:CLOCK:BMAL1:E-box complex. (C) New model for the mammalian circadian clock. The figure shows a semiquantitative heat map representation of CRY1 and PER2 protein expression as well as the ChIP data for CLOCK:BMAL1 and CRY1 over a circadian cycle and its consequences with regard to interactions of core clock proteins with the E-box and the effects of these interactions on transcription of genes (Nr1d1 and Dbp) regulated exclusively by the core TTFL.

The circadian clock is the internal timekeeping system that controls cyclic changes in physiology and behavior to prepare the organism for the unique challenges of the solar day.  In mice and humans the circadian rhythm at the organismal level is generated by a molecular clock of periodicity of ~24 hrs.  The molecular clock consists of a transcription-translation feedback loop (TTFL) in which the heterodimeric transcriptional activator CLOCK-BMAL1 promotes transcription of transcriptional repressors, CRY (Cryptochrome) and PER (Period) which counter the activity of CLOCK-BMAL1 as shown in Fig. 3AOur group discovered that CRY is a core human clock protein and that it mediates repression by two mechanisms (Fig. 3B,C): In one CRY binds to the CLOCK-BMAL1 complex on DNA and blocks its interaction with the transcription machinery.  In the second mode of repression, the co-repressor PER displaces the entire activator complex from the promoter in a CRY-dependent manner.  Our research has provided a mechanistic basic for how this dual repression mechanism confers precision and resilience and, at the same time, flexibility and adaptability to the signaling pathways and networks and therefore influences physio-pathologic conditions ranging from sleep regulation and jetlag to metabolic syndrome to cancer.  We discovered that the circadian clock regulates nucleotide excision repair and hence the susceptibility to UV-induced skin cancer as a function of time of the day of exposure to light.  Because excision repair is also the main repair mechanism for removing DNA lesions from the genome that are generated by the anti-cancer drug cisplatin, we are currently working at translating our finding of clock-excision repair connection to develop improved chemotherapy regimens.

RECENT PUBLICATIONS pubmed.png(Click for full publication list)

  • Tan C, Liu Z, Li J, Guo X, Wang L, Sancar A, Zhong D. (2015) The molecular origin of high DNA-repair efficiency by photolyase. Nat Commun. 6:7302. 
  • Hu J, Adar S, Selby CP, Lieb JD, Sancar A. (2015) Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution. Genes Dev. 29(9):948-60.
  • Kemp MG, Lindsey-Boltz LA, Sancar A. (2015) UV light potentiates STING (stimulator of interferon genes)-dependent innate immune signaling through deregulation of ULK1 (Unc51-like kinase 1). J Biol Chem. 290(19):12184-94.
  • Lindsey-Boltz LA, Kemp MG, Capp C, Sancar A. (2015) RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling. Cell Cycle 14(1):99-108.
  • Gaddameedhi S, Selby CP, Kemp MG, Ye R, Sancar A. (2014) The Circadian Clock Controls Sunburn Apoptosis and Erythema in Mouse Skin. J Invest Dermatol. 135(4):1119-27.
  • Sancar A, Lindsey-Boltz LA, Gaddameedhi S, Selby CP, Ye R, Chiou Y-Y, Kemp MG, Hu J, Lee JH, Ozturk N. (2015) Circadian Clock, Cancer, and Chemotherapy. Biochemistry 54(2):110-23.
  • Ye R, Selby CP, Chiou YY, Ozkan-Dagliyan I, Gaddameedhi S, Sancar A. (2014) Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock. Genes Dev. 28(18):1989-98.
  • Kemp MG, Gaddameedhi S, Choi JH, Hu J, Sancar A. (2014) DNA repair synthesis and ligation affect the processing of excised oligonucleotides generated by human nucleotide excision repair. J Biol Chem. 289(38):26574-83.
  • Tan C, Guo L, Ai Y, Li J, Wang L, Sancar A, Luo Y, Zhong D. (2014) Direct Determination of Resonance Energy Transfer in Photolyase: Structural Alignment for the Functional State. J Phys Chem A. 118(45):10522-30.
  • Ozturk N, Selby CP, Zhong D, Sancar A. (2014) Mechanism of Photosignaling by Drosophila Cryptochrome:Role of the Redox Status of the Flavin Chromophore. J Biol Chem. 289: 4634-4642.
  • Annayev Y, Adar S, Chiou YY, Lieb J, Sancar A, Ye R. (2014) Gene Model 129 (Gm129) Encodes a Novel Transcriptional Repressor that Modulates Circadian Gene Expression. J Biol Chem. 289: 5013-5024.
  • Lindsey-Boltz LA, Kemp MG, Reardon JT, Derocco V, Iyer RR, Modrich P, Sancar A. (2014) Coupling of Human DNA Excision Repair and ATR-mediated DNA Damage Checkpoint in a Defined In Vitro System. J Biol Chem. 289: 5074-5082.
  • Choi JH, Gaddameedhi S, Kim SY, Hu J, Kemp MG, Sancar A.  (2014) Highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells. Nucleic Acids Res. 42(4):e29.
  • Liu Z, Tan C, Guo X, Li J, Wang L, Sancar A, Zhong D. (2013) Determining complete electron flow in the cofactor photoreduction of oxidized photolyase.  Proc Natl Acad Sci U S A 110(32):12966-71.
  • Liu Z, Zhang M, Guo X, Tan C, Li J, Wang L, Sancar A, Zhong D. (2013) Dynamic determination of the functional state in photolyase and the implication for cryptochrome. Proc Natl Acad Sci U S A 110(32):12972-7.
  • Ozkan-Dagliyan I, Chiou YY, Ye R, Hassan BH, Ozturk N, Sancar A. (2013) Formation of Arabidopsis Cryptochrome 2 Photobodies in Mammalian Nuclei: Application as an Optogenetic DNA Damage Checkpoint Switch. J Biol Chem. 288(32):23244-51.
  • Hu J, Choi JH, Gaddameedhi S, Kemp MG, Reardon JT, Sancar A. (2013) Nucleotide Excision Repair in Human Cells: Fate of the Excised Oligonucleotide Carrying DNA Damage In Vivo. J Biol Chem. 288(29):20918-26.  
  • Hassan BH, Lindsey-Boltz LA, Kemp MG, Sancar A. (2013) Direct role for the replication protein Treslin (Ticrr) in the ATR-mediated checkpoint response. J Biol Chem. 288(26):18903-10.
  • Ozturk N, VanVickle-Chavez SJ, Akileswaran L, Van Gelder RN, Sancar A. (2013) Ramshackle (Brwd3) promotes light-induced ubiquitylation of Drosophila Cryptochrome by DDB1-CUL4-ROC1 E3 ligase complex. Proc Natl Acad Sci U S A 110(13):4980-5.
  • Lee JH, Gaddameedhi S, Ozturk N, Ye R, Sancar A. (2013) DNA Damage-Specific Control of Cell Death by Cryptochrome in p53-Mutant Ras-Transformed Cells. Cancer Res. 73(2):785-91.

Lab Contact: 

Lab Rooms: 3073 Genetic Medicine Building
Lab Phone: 919-962-0115
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