Welcome to the Expression and Molecular Biology (EMB) Core. The EMB Core consists of both service and research components to support the NIH/NCI-funded Structural Cell Biology of DNA Repair Machines (SBDR) Program Project. The SBDR Program, centered at Berkeley Lab and led by Drs. John Tainer and Priscilla Cooper, is a multi-investigator (16), multi-institutional (12), interdisciplinary effort optimized for the extreme challenges of characterizing transient complexes and dynamic conformations acting in DNA damage responses by combining individual investigator labs into a trans-disciplinary effort. The program seeks to integrate knowledge of DNA repair proteins and pathways into physiological models of DNA repair by characterizing keystone complexes functioning as regulatory nodes in complex cellular responses to DNA damage. To define conformational switching and assembly for DR machines, the program applies macromolecular X-ray crystallography (MX) for structural detail in combination with small angle X-ray scattering (SAXS) and NMR for conformation and assembly in solution. Our ability to couple structural results with biochemical, genetic, and cellular studies provides an opportunity to characterize DNA repair proteins and their biologically relevant interactions, complexes, and conformations: information that will elucidate mechanisms of cancer etiology and ultimately lead to improving therapeutic outcomes.
Under the directorship of Dr. Cooper, the goals of the EMB Core are to:
- engineer vectors for high-level expression of soluble target proteins and protein complexes,
- provide sufficient amounts of proteins and complexes, or cells expressing target proteins and complexes to the SBDR projects for structural and functional characterization,
- identify and characterize protein interactions, and
- establish and maintain a repository of reagents for the SBDR program.
DNA Cloning. The EMB Core utilizes a variety of expression vectors for analysis of protein expression in bacteria, insect cells, and possibly in yeast, as well as for functional studies in mammalian cells. The EMB Core can accommodate custom cloning into the vector of choice as an untagged, tagged, or cleavable-tagged protein. We place priority on engineering vectors or employing expression systems that allow co-expression of protein complexes in a soluble form for functional and structural studies. Commercial and custom dual expression vectors are available for the comparative expression of target genes in bacteria and insect cells. In addition, custom bacterial polycistronic and polypromoter vectors are available upon request. We use QuickChange mutagenesis (Agilent) to generate point mutation, deletion or insertion for gene of interest.
Protein Expression. Depending on the nature of the protein and project, expression of a target gene can be tested in bacteria and insect cells. We have streamlined standard procedures for optimizing soluble expression in bacteria and insect cells. The EMB Core has the capacity to scale up expression of target proteins or protein complexes in bacteria (up to 6 L per week) and insect cells (up to 8 L per week) to facilitate high yields of recombinant protein purification by the SBDR member laboratories.
Protein Interactions. We use affinity co-purification in solution, including GST, MBP, Ni-NTA, HisPur Co, and Flag pull-downs, to determine protein complex formation and map protein interacting domains. The yeast-two-hybrid (Y2H) interaction study is available upon request. We also complement these studies using antibody co-IP and Far Western analysis. Protein/protein and protein/DNA interactions can be measured quantitatively using the Biacore 3000, an automated biosensor capable of measuring interactions between molecules immobilized on the surface of a chip (ligands) and molecules in solution (analytes). See GE healthcare website for details. We are also equiped with an AKTA micro system that allows analytic scale separation by size exclusion column to validate the formation of protein complexes.
Protein Purification. The EMB Core is equipped with Bio-Rad Biologic LP system and GE AKTA FPLC for small- (25~500 ml cells) and pilot-scale (up to 2 L cells) purification of recombinant proteins from bacteria and insect cells. We routinely perform small-scale, one-step affinity batch purification to verify the protein tag in the construct design. Pilot-scale purification combined with native gel analysis assists in screening and identifying suitable structural target protein fragments and in guiding construct design. Pilot-scale purified recombinant proteins are available for biochemical studies, functional assays, and domain mapping. We also provide purified recombinant proteins of EM-, NMR-, and SAXS-quality for structural analysis by SBDR labs.
Repository of Reagents. The EMB Core is continuing to maintain a repository of purified proteins, antibodies, recombinant expression constructs, bacterial host strains, and expression vectors for distribution to SBDR laboratories. A series of commonly used expression vectors and recombinant constructs for expression of DNA repair proteins have been purified, sequenced and catalogued along with various bacterial host strains. Groups across the SBDR program can access the repository reagents upon request. Reagents generated through individual lab collaborations can be distributed upon consent of the specific laboratory.
Longerich S, Kwon YH, Tsai MS, Hlaing AS, Kupfer G, and Sung P. Regulation of FANCD2 and FANCI monoubiquitination by their interaction and by DNA. Nucleic Acids Res. In press (2014).
Zhao Q, Saro D, Sachpatzidis A, Singh TR, Schlingman D, Zheng XF, Mack A, Tsai MS, Mochrie S, Regan L, Meetei AR, Sung P, Xiong Y. The MHF complex senses branched DNA by binding a pair of crossover DNA duplexes. Nat Commun. 5, 2987 (2014).
Shell SM, Hawkins EK, Tsai MS, Hlaing AS, Rizzo CJ, Chazin WJ. Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA. DNA Repair (Amst). 12, 947-53 (2013).
Della-Maria J, Hegde ML, McNeil DR, Matsumoto Y, Tsai MS, Ellenberger T, Wilson DM, Mitra S, and Tomkinson AE. The interaction between polynucleotide kinase phosphatase and the DNA repair protein XRCC1 is critical for repair of DNA alkylation damage and stable association at DNA damage sites. J Biol Chem. 287, 39233-39244 (2012).
Querol-Audi J, Yan C, Xu X, Tsutakawa SE, Tsai MS, Tainer JA, Cooper PK, Nogales E, and Ivanov I. Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability. Proc Natl Acad Sci U S A. 109, 8528-8533 (2012).
Dunlop MH, Dray E, Zhao W, San Filippo J, Tsai MS, Leugn SG, Schild D, Wiese C, and Sung P. Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair. J Biol Chem. 287, 12343-12347 (2012).
Dunlop MH, Dray E, Zhao W, Tsai MS, Wiese C, Schild D, and Sung P. RAD51-associated protein 1 (RAD51AP1) interacts with the meiotic recombinase MDC1 through a conserved motif. J Biol Chem. 286, 37328-37334 (2011).
Della-Maria J, Zhou YI, Tsai MS, Kuhnleln J, Carney JP, Paull TT, and Tomkinson AE. Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway. J Biol Chem. 286, 33845-33853 (2011).
Trego KS, Chernikova SB, Davalos AR, Perry JJ, Finger LD, Ng C, Tsai MS, Yannone SM, Tainer JA, Campisi J, Cooper PK. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defected in Werner syndrome. Cell Cycle. 10, 1998-2007 (2011).
Dray E, Dunlop MH, Kauppi L, Filippo JS, Wiese C, Tsai MS, Begovic S, Schild D, Jasin M, Keeney S, and Sung P. Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1). Proc Natl Acad Sci U S A. 108, 3560-3565 (2011).
Dray E, Etchin J, Wiese C, Saro D, Williams GJ, Hammel M, Yu X, Galkin VE, Liu D, Tsai MS, Sy SMH, Schild D, Egelman E, Chen J. & Sung P. Enhancement of the RAD51 recombinase by the tumor suppressor PALB2. Nat Struct Mol Biol. 17, 1255-1259 (2010).
Iyer RR, Pluciennik A, Genschel J, Tsai MS, Beese LS, and Modrich P. MutLalpha and PCNA share binding sites on MutSbeta. J Biol Chem. 285, 11730-11739 (2010).
Chen X, Ballin JD, Della-Maria J, Tsai MS, White EJ, Tomkinson AE & Wilson GM. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair. DNA Repair (Amst). 8, 961-968 (2009).
Wiese C, Dray E, Groesser T, San Filippo J, Shi I, Collins DW, Tsai MS, Williams GJ, Rydberg B., Sung P. & Schild D. Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement. Mol Cell. 28, 482-490 (2007).
Bugni JM, Han J, Tsai MS, Hunter DJ & Samson LD. Genetic association and functional studies of major polymorphic variants of MGMT. DNA Repair (Amst). 6, 1116-1126 (2007).
EMB Core Acknowledged by SBDR Publications
Couch FB, Bansbach CE, Driscoll R, Luzwick JW, Glick GG, Betous R, Carroll CM, Jung SY, Qin J, Cimprich KA, and Cortez D. ATR phosphorylates SMARCAL1 to prevent replication fork collapse. Gene Dev. 27, 1610-1623 (2013).
Hedge ML, Hedge PM, Bellot LJ, Mandal SM, Hazra TK, Li G-M, Boldogh I, Tomkinson AE, and Mitra S. Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc Natl Acad Sci USA. E 3090-3099 (2013).Tseng Q, Orans J, Hast MA, Iyer RR, Changela A, Modrich PL, and Beese LS. Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSbeta. Acta Cryst. F67, 947-952 (2011)
Orans J, McSweeney EA, Iyer RR, Hast MA, Hellinga HW, Modrich P, and Beese LS. Structures of human exonuclease I DNA complexes suggest a unified mechanism for nuclease family. Cell. 145, 212-223 (2011).
Banerjee D, Mandal SM, Das A, Hegde ML, Das S, Bhakat KK, Boldogh I, Sarkar PS, Mitra S, and Hazra TK. Preferential repair of oxidized base damage in the transcribed genes of mammalian cells. J Biol Chem. 286, 6006-6016 (2011).
Volunteer research student assistants.
Contact EMB Core Head Miaw-Sheue Tsai for information.
Beese, Lorena (Duke University), 2005 – present
Carney, Jim (University of Maryland), 2005
Chazin, Walter (Vanderbilt University), 2008 – present
Chen, David (University of Texas Southwestern), 2002 – present
Cooper, Priscilla K. (Berkeley lab), 2002 – present
Cortez, David ((Vanderbilt University), 2011 – present
Ellenberger, Tom (Washington State University, St. Louise), 2002 – present
Glover, Mark (University of Alberta), 2009 – present
Kolodner, Richard (UCSD), 2005 & 2010
Kuriyan, John (University of California, Berkeley) 2007
Matsumoto, Yoshihiro (Fox Chase Cancer Center), 2003 – 2009
McMurray, Cynthia (Berkeley Lab), 2009 – present
Modrich, Paul (Duke University), 2005 – present
Mitra, Sankar (University of Texas Medical Branch), 2003 – 2009; 2012 – present
Nogales, Eva (UC Berkeley), 2008 – 2013
Paull, Tanya (University of Texas, Austin), 2006 – 2007
Qin, Jun (Baylor College of Medicine), 2004 – 2005
Samson, Leona (Massachusetts Institute of Technology), 2006 – 2007
Schärer, Orlando (New York State University, Stony Brook), 2008 – present
Schild, David (Berkeley Lab), 2003 – present
Sung, Patrick (Yale University), 2006 – present
Tainer, John (Berkeley Lab), 2003 – present
Thompson, Larry (Lawrence Livermore National Laboratory), 2003
Tomkinson, Alan (University of New Mexico Cancer Center), 2006 – present
Wyman, Claire (Erasmus University Medical Center), 2007
* Honors students
* * Research assistants / associates
Schild, David (Berkeley Lab, Co-Director, 2001-2006)
Berardini, Mark (Berkeley Lab, Head, 2002-2005)
Han, Zsu-Min (Summer 2002)
Ching, Maggie (Fall 2002 – Spring 2004)
Neupane, Rupak (Fall 2002 – Summer 2003)
Qi, Jane (Fall 2002 – Spring 2004)
Win, Khine Zar (Fall 2003 – Spring 2005) *
Cho, May Thet (Fall 2003 – Spring 2005) *
Ceja, Jorge (Summer 2004 – Fall 2005)
Calderwood, Montgomery (Summer – Fall 2004)
Lin, Stephanie (Summer 2004)
Win, Ei Thandar (Summer 2004)
Khine, Ellise (Summer 2004)
Shu, Yu Yu (Summer 2004)
Yuen, Kevin (January 2005)
Kim, Tiffany (Spring 2005 – Summer 2007) *
Zhang, Ying (Spring 2005)
Chin, Eugene (Spring 2005 – Spring 2006) *
Alonso, James (Spring 2005)
Barthure, Archana (Summer 2005)
Ng, Cliff (2005 – 2010) **
Cooper, Brian (Summer 2005 – Spring 2006) **
Lai, Joyce (Fall 2005 – Spring 2006; Spring 2007)
Yang, Nancy (Fall 2005 fall – Spring 2007) *
Sun, Melody (Spring 2006 – Summer 2007)
Kao, Jessica (Spring 2006 – Spring 2007)
Li, Patricia (Spring 2006 – Spring 2007) **
Read, Avilla (Summer 2006, high school intern)
Thompson, Russell (Summer 2006, 2007, 2008, HHSRPP high school intern)
Moitoza, Jennifer (Fall 2006 – Spring 2007; Summer 2007 – Spring 2008) **
Tet, Connie (Fall 2006 – Summer 2007)
Pollard, Lance (Fall 2006 – Spring 2007)
Akbar, Ahmed (Summer 2007 – Spring 2009)
Din, Richard (Fall 2007)
Wang, Shou-Ping (Pam) (Fall 2007 – Summer 2008) **
Chan, Odilia (Fall 2007 – Spring 2009)
Hlaing, Aye Su (Spring 2008 – Spring 2009; Summer 2009 – Summer 2012)**
Aung, Phyo (Spring 2008 – Fall 2008; Summer 2009 – Summer 2011) **
Khatri, Krish (Summer 2008 – Summer 2009) **
Chou, Austin (Summer 2008 – Summer 2009)
Nguyen, Khanh (Summer 2008 – Spring 2010) **
Huynh, Stephen (Summer 2008 – Spring 2009; Spring 2010)
Lee, Janelle (Fall 2008)
Quach, Joanna (Summer 2009, BLIPS high school intern)
Yee, Andrew (Summer 2009)
Lin, Hao-Nan (Tom) (Fall 2009 – Spring 2010)
Ge, Lu (Fall 2009 – Summer 2010)
Shih, Brian (Fall 2009 fall – Fall 2011)
Lin, Connie (Summer 2010, CCI intern; Spring – Summer 2011)
Ouyang, An-Chieh (Summer 2010)
Chan, Vincent (Summer 2010)
Hsu, Shelly (Summer 2010 – Spring 2011)
Jiang, Shuai (Summer 2010 – Spring 2011)
Ow, Jennifer (Fall 2010 – Spring 2012)
Magana, Ramsey (Spring 2011 – Spring 2012)
Peralta Betancourt, Alvin (Summer 2011, SULI intern)
Cortes, Adrian (Summer 2011, CCI intern; Summer 2012, SULI intern; Fall 2013, SULI intern)
Dalida, Janice (Fall 2011 – Spring 2012; Summer 2012, SULI intern)
Veres, Robert (Summer 2012, high school intern)
Pia, Shermila (Aug-Oct 2012)
Pravdic, Masa (June 2012 – May 2013)
Doghmi, Myriam (May – Aug 2013, Summer intern)
Douglas, Marquis (June – Aug 2013, SULI intern)