Research

Genomic instability is a hallmark of cancer. Somatic genetic instability, leading to DNA translocations, gross insertions, deletions and duplications, reshapes cancer genomes and creates gene fusions that can increase oncogenic potential and/or support tumor progression. Key to formation of these chromosomal aberrations are DNA breaks followed by error-generating break repair processing, which may join two noncontiguous segments of a chromosome (deletions), insert novel sequences (insertions), or fuse two different chromosomes (translocations).

We have elucidated structures and mechanisms for three major DNA repair pathways currently known to act upon DNA double-strand breaks (DSBs): (i) non-homologous end joining (NHEJ), which is active throughout the cell cycle and does not require sequence homology; (ii) homology dependent repair (HDR), which is active in S phase and G2 and uses homologous sequences to restore chromosome continuity without causing errors or cancer instability, and (iii) alternative-end-joining (Alt-EJ), which employs microhomology to allow error prone repair for stalled replication and at dsDNA breaks (DSBs). Sequence analyses of whole cancer genomes plus the sequence contexts at the points of DSB fusion and the finding that HDR is often compromised in cancer cells, indicates that somatic chromosomal aberrations involve DNA repair pathways that play minor or back-up roles in normal cells. Consistent with this notion is the observation that the HDR-deficient genetic signature noted in many breast cancers correlates strongly with >3 bp insertions and deletions. This, together with the presence of overlapping microhomologies at the breakpoints, is consistent with Alt-EJ and points to its increased role for replication-based mechanisms of DNA repair in cancer cells. Recently we found that XRCC1 partners with the MRE11 nuclease and Pol Q to promote replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells.

Loss of replication fidelity appears to play a pivotal role in cancer-related genomic instability, although tissue-specific mechanisms including transcription-associated replication tress, are also involved. In this context, we examined damage resulting from DNA sequences with the potential to fold into non-B DNA structures (PONDS). We discovered an association between PONDS-forming repeats and breakpoints that is further supported by the finding that rearrangements tend to recur at near identical genomic positions in different patient and tumor samples. These data suggest that sequences with the potential to fold into non-B DNA structures are an intrinsic risk factor for the occurrence of translocations and deletions in cancer genomes.

Replication forks stall due to oncogenic and other stress factors, resulting in fork restart or collapse and consequent genome instability or cell death. Thus, current cancer therapeutics are motivated in part by targeting replication through crosslinking agents, topoisomerase inhibitors and high-dose radiation. However, other mechanisms that lead to replication arrest have emerged, including head-on collision with transcription and unresolved DNA secondary structures, commonly referred to as non-B DNA. We therefore examined the possibility that non-B DNA can form in chromosomal DNA, further block ingreplication and causing genomic instability in cancer. We performed an unbiased analysis of translocation breakpoints mapped at single base-pair resolution in human cancer genomes with the occurrence of five types of PONDS-forming repeats (direct repeats, inverted repeats, homo(purine•pyrimidine) tracts with mirror repeat symmetry, alternating purine-pyrimidine runs, and G-quartets), which may form slipped structures, hairpin/cruciforms, triplex (H-DNA), left-handed Z-DNA, and quadruplex DNA (G4-DNA), respectively. Strikingly, we found a strong correlation between PONDS-forming repeats and rearrangement breakpoints, particularly for translocations.

We furthermore examined common fragile sites (CFSs) as difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. Notably, CSFs are hotspots for chromosomal instability in cancer. Trans-lesion synthesis polymerases, such as Pol eta, can efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability. We found an increase in fork pausing at fibroblast-specific CFSs in Pol eta-deficient fibroblasts. Importantly, pause sites were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. By examining single-nucleotide polymorphisms (SNPs) genome wide, we found that SNPs at pause sites occurred more frequently than expected based on their sequence composition and at rates only seen at the highest end of the spectrum genome-wide. Our combined findings suggest Pol eta replicating through CFSs acts in genetic variations found in the human population at these sites.

Exemplary Publications:

1. Bacolla A, Tainer JA, Vasquez KM, Cooper DN. Translocation and deletion breakpoints in cancer genomes are associated with potential non-B DNA-forming sequences. Nucleic Acids Res. 44:5673-88, 2016.
2. Thapar R, Bacolla A, Oyeniran C, Brickner JR, Chinnam NB, Mosammaparast N, Tainer JA. RNA Modifications: Reversal Mechanisms and Cancer. Biochemistry. 58:312-329, 2019.
3. Bacolla A, Ye Z, Ahmed Z, Tainer JA. Cancer mutational burden is shaped by G4 DNA, replication stress and mitochondrial dysfunction. Prog Biophys Mol Biol. 147:47-61, 2019.
4. Singh M, Bacolla A, Chaudhary S, Hunt CR, Pandita S, Chauhan R, Gupta A, Tainer JA, Pandita TK. Histone Acetyltransferase MOF Orchestrates Outcomes at the Crossroad of Oncogenesis, DNA Damage Response, Proliferation, and Stem Cell Development. Mol Cell Biol. 40:e00232-20, 2020.
5. Eckelmann BJ, Bacolla A, Wang H, Ye Z, Guerrero EN, Jiang W, El-Zein R, Hegde ML, Tomkinson AE, Tainer JA, Mitra S.  XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells. NAR Cancer. 2: zcaa013, 2020.
6. Lees-Miller JP, Cobban A, Katsonis P, Bacolla A, Tsutakawa SE, Hammel M, Meek K, Anderson DW, Lichtarge O, Tainer JA, Lees-Miller SP. Uncovering DNA-PKcs ancient phylogeny, unique sequence motifs and insights for human disease. Prog Biophys Mol Biol. 163:87-108, 2021.
7. Berroyer A, Bacolla A, Tainer JA, Kim N. Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability. Microb Cell. 9:52-68, 2022.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other DNA glycosylases (DGs) of the base excision repair (BER) pathway. Yet, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remained enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super resolution microscopy and co-immunoprecipitation assays, we found that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) revealed nonrandom AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Thus, DNA repair proteins can be pre-targeted to damage susceptible sites.

In general, we have discovered multiple fundamental mechanisms of DNA damage recognition and removal. We found that base repair enzymes flip nucleotides from dsDNA double helix into protein pockets for specific recognition. Our enzyme-DNA complex structures defined the basis for damage specificity and repair for multiple pathways essential to genomic integrity: human uracil-DNA glycosylase, APE1 (the central abasic site nuclease in base repair), and alkyl-guanine transferase, a direct damage reversal protein for alkylation damage and resistance factor for chemotherapy. We solved human ABH3 (hABH3) structures in complex with iron and 2-oxoglutarate (2OG), defined key site-directed mutants, and established hABH3 as a member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couple substrate oxidation to conversion of 2OG into succinate and CO2. We identified and named the helix-hairpin-helix (HhH) motif and its roles in protein and in sequence-independent nucleic acid binding. We revealed mimicry in DNA repair regulation and how to look for protein mimicry of DNA in biological pathways. We solved multiple exemplary structures of Fe-S cluster-containing DNA repair enzymes: MutY, endonuclease III, XPD, and EXO5. Prior to PARP inhibitors, we pioneered cancer-relevant targeting of the DNA damage response in our work on MGMT/AGT inhibitor.

Our XPG (ERCC5) nuclease and XPD (ERCC2) helicase structures showed how single site mutations can cause different diseases: cancer pre-dispositions, aging, and developmental failure after birth. Together our XPG and XPD structures and analyses provided key benchmarks to develop actionable knowledge for variants of unknown significance (VUS). We developed and applied computational and experimental methods to make and characterize multi-protein TFIIH complexes and their functional movements. We co-discovered and named SUMO-targeted ubiquitin ligases (STUbLs) that join the ubiquitin and SUMO pathways and act in genome stability. For the replication-repair interface, we solved structures of FEN1 in combination with DNA and PCNA peptides, and discovered the ATR-regulated fork restart nuclease EXO5.

For HDR, we determined how ATP-driven conformations of RAD50 control end joining versus excision by MRE11. Our foundational results on RAD50 break repair ATPase defined the first correct ABC ATPase assembly and the basis for ATP-recognition by the signature motif. Our structures formed the prototype for ABC ATPases including the ABC transporters and CFTR. Our structures of Mre11 break repair nuclease in complex with DNA revealed how Mre11 can function differently to process replication forks and DNA double strand breaks. We identified and solved structures for the Rad50 zinc-hook showing how it keeps DNA double strand breaks from becoming chromosome breaks by joining Mre11-Rad50 complexes together across the break. We determined Nbs1 DNA break repair sensor structures, showed how it recognizes its phosphor-protein partners, and uncovered how flexibility and plastic deformations create allosteric signals from the DNA break to the Mre11-Rad50 core complex. Our PTIP homolog domain structures bound to gammaH2AX peptide linked nucleosomes to repair sites. With Susan Lees-Miller, we defined dynamic non-homologous end joining (NHEJ) complexes for human dsDNA break repair including the role of a scaffolding and phase condensate role for long non-coding RNA.

Based upon our structurally-defined conformational changes acting in DNA repair mechanisms, we are developing new allosteric inhibitors to target conformations and assemblies rather than design transition state analogues as inhibitors for base repair enzymes UDG and the PAR glycohydrolase PARG.

Exemplary Publications:

1. Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, Tainer JA. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell. 101:789-800, 2000.
2. Fan L, Fuss JO, Cheng QJ, Arvai AS, Hammel M, Roberts VA, Cooper PK, Tainer JA. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell. 133:789-800, 2008.
3. Williams RS, Dodson GE, Limbo O, Yamada Y, Williams JS, Guenther G, Classen S, Glover JN, Iwasaki H, Russell P, Tainer JA. Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand break processing and repair. Cell. 139: 87-99, 2009.
4. Heideker J, Prudden J, Perry JJ, Tainer JA, Boddy MN. SUMO-targeted ubiquitin ligase, Rad60, and Nse2 SUMO ligase suppress spontaneous Top1-mediated DNA damage and genome instability. PLoS Genet. 7:e1001320, 2011.
5. Shibata A, Moiani D, Arvai AS, Perry J, Harding SM, Genois MM, Maity R, van Rossum-Fikkert S, Kertokalio A, Romoli F, Ismail A, Ismalaj E, Petricci E, Neale MJ, Bristow RG, Masson JY, Wyman C, Jeggo PA, Tainer JA. DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. Mol. Cell. 53:7-18, 2014.
6. Syed A, Tainer JA. The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair. Annu Rev Biochem. 87:263-294, 2018.
7. Thapar R, Wang JL, Hammel M, Ye R, Liang K, Sun C, Hnizda A, Liang S, Maw SS, Lee L, Villarreal H, Forrester I, Fang S, Tsai MS, Blundell TL, Davis AJ, Lin C, Lees-Miller SP, Strick TR, Tainer JA. Mechanism of efficient double-strand break repair by a long non-coding RNA. Nucleic Acids Res. 48:10953-10972, 2020.
8. Tsutakawa SE, Sarker AH, Ng C, Arvai AS, Shin DS, Shih B, Jiang S, Thwin AC, Tsai MS, Willcox A, Her MZ, Trego KS, Raetz AG, Rosenberg D, Bacolla A, Hammel M, Griffith JD, Cooper PK, Tainer JA. Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations. Proc Natl Acad Sci USA. 117:14127-14138, 2020.
9. Ye Z, Xu S, Shi Y, Bacolla A, Syed A, Moiani D, Tsai CL, Shen Q, Peng G, Leonard PG, Jones DE, Wang B, Tainer JA*, Ahmed Z. GRB2 enforces homology-directed repair initiation by MRE11. Science Adv. 7(32):eabe9254, 2021. *co-corresponding author
10. Bacolla A, Sengupta S, Ye Z, Yang C, Mitra J, De-Paula RB, Hegde ML, Ahmed Z, Mort M, Cooper DN, Mitra S, Tainer JA. Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin. Nucleic Acids Res. 49: 221-243, 2021.
11. Tsutakawa SE, Bacolla A, Katsonis P, Bralic A, Hamdan SM, Lichtarge O, Tainer JA, Tsai CL. Decoding Cancer Variants of Unknown Significance for Helicase-Nuclease-RPA Complexes Orchestrating DNA Repair During Transcription and Replication. Front Mol Biosci. 8:791792, 2021
12. Hambarde S, Tsai CL, Pandita RK, Bacolla A, Maitra A, Charaka V, Hunt CR, Kumar R, Limbo O, Le Meur R, Chazin WJ, Tsutakawa SE, Russell P, Schlacher K, Pandita TK, Tainer JA. EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart. Mol Cell . 81: 2989-3006.e9, 2021.

We have provided breakthrough results on structural mechanisms for the control of reactive oxygen species (ROS), DNA damage and immune responses. The responses to reactive oxygen, DNA damage (DDR) and immune stress are organized networks of multiple interwoven components evolved to enable homeostasis, limit damage and maintain genome fidelity. We are examining their functional interplay. ROS are enzyme intermediates and signals that can also act as cytotoxins to kill infected and cancer cells and provide tools for biotechnology. In aerobic cells, radicals are scavenged by oxygen to superoxide, so superoxide dismutases (SODs) and their partner catalase (CAT) are master ROS regulators. Thus, SOD and CAT are essential to life and genome integrity, and more specifically for oxidative homeostasis and host immune response to cancer. We determined first structures of human cytoplasmic Cu,Zn SOD and human mitochondrial Mn SOD. We developed a leading unified theory for how the fatal neurodegenerative disease amyotrophic lateral sclerosis is caused by single-site SOD mutations. We solved human catalase and glutathione S-transferase structures to inform their mechanisms and inhibition. We defined the first structures of nitric oxidize synthases and defined mechanisms for NO synthesis and control as a signal and defensive cytotoxin.

With Carolyn Bertozzi, we provided the breakthrough structure of the formylglycine-generating enzyme (FGE) that has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. We discovered FGE acts via O2 activation at an unexpected Cu(I) site in the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. These collective results provided a detailed mechanistic framework for understanding why nature chose this structurally unique mono-copper active site to catalyze oxidase chemistry for sulfatase activation. Overall, our structures, data and concepts have resolved long-standing problems and provided the framework for ongoing research in ROS control enzymes, inflammation and innate immunity.

As antioxidants, Vitamin E (VitE) supplements have been implicated in strengthening immunity with anti-inflammatory, anti-atherosclerotic and antitumor effects in animal models. Recently, with Dihua Yu, we examined mechanisms underlying the disease-modifying activities of VitE and found that VitE boosted antitumor immunity by mainly enhancing the tumor antigen presentation of dendritic cells (DCs) and DC-derived extracellular vesicles (DC-EVs) by inhibiting the Src homologous-region-2-domain-containing phosphatase-1 (SHP1), a DC intrinsic checkpoint. Compellingly, unleashing DCs and DC-EVs by VitE is an immediately applicable strategy for inducing efficient antitumor immunity and enhancing immunotherapy response.

With Mein-Chie Hung, we helped show that the antibiotics daunorubicin, doxorubicin, epirubicin and actinomycin D induce the critical immune-checkpoint programmed death ligand1 (nPD-L1) and gasdermin C (GSDMC) expression as well as caspase-8 activation, leading to pyroptosis in cancer cells. This implies that these drugs may promote anti-tumor immunity in the treatment of PD-L1+ or GSDMC+ breast tumors.

We were the first to show that anti-peptide antibodies better recognize flexible protein regions, a discovery still used for vaccine and antibody design. For pathogen virulence factors, we solved the first and only full-length structures of the flexible, fiber-forming and membrane protein pilin. We elucidated its assembly into the bacterial pilus informing pathogen mobility, adherence, recombination and escape from the immune system. We then defined multiple analogous archaeal flagella mobility components and the assembly of archaeal motility machinery. We solved key structures of bacterial toxins and identified the novel recognition motif of Rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum along with structural insights for its functional motif relevant to pathogenicity and DNA damage responses. We are now applying our PARG project expertise to inhibit the COVID-19 macrodomain acting in viral replication and immune suppression.

Overall, we are interested in how ROS and the DNA damage response (DDR) shape both innate and adaptive immune pathways in the context of: (i) innate immunity, where DDR components mainly enhance cytosolic DNA sensing and its downstream STimulator of INterferon Genes (STING)-dependent signaling; (ii) adaptive immunity, where the DDR is needed for the assembly and diversification of antigen receptor genes that is requisite for T and B lymphocyte development. Imbalances between DNA damage and repair impair tissue homeostasis and lead to replication and transcription stress, ROS, mutation accumulation and even cell death. These impacts from DDR defects can then drive tumorigenesis, secretion of inflammatory cytokines and aberrant immune responses. Yet, DDR deficiency or inhibition can also directly enhance innate immune responses. Furthermore, DDR defects plus the higher mutation load in tumor cells synergistically produce primarily tumor-specific neoantigens, which are powerfully targeted in cancer immunotherapy by employing immune checkpoint inhibitors to amplify immune responses. Thus, elucidating DDR-immune response interplay may provide critical connections for harnessing immunomodulatory effects plus targeted inhibition to improve efficacy of radiation and chemotherapies, of immune checkpoint blockade and of combined therapeutic strategies.

We are continuing to define and test underlying mechanisms for the interplay of dynamic reactive oxygen, DNA damage and immune responses with their links to inflammation, pathogenesis, cell death and cancer.

Exemplary Publications:

1. Parge HE, Forest KT, Hickey MJ, Christensen DA, Getzoff ED, Tainer JA. Structure of the fibre-forming protein pilin at 2.6 A resolution. Nature. 378:32-38, 1995.
2. Crane BR, Arvai AS, Ghosh DK, Wu C, Getzoff ED, Stuehr DJ, Tainer JA. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science. 279:2121-2126, 1998.
3. Liu X, Hammel M, He Y, Tainer JA, Jeng US, Zhang L, Wang S, Wang X. Structural insights into the interaction of IL-33 with its receptors. Proc Natl Acad Sci U S A. 110:14918-23, 2013.
4. Reindl S, Ghosh A, Williams GJ, Lassak K, Neiner T, Henche AL, Albers SV, Tainer JA. Insights into FlaI functions in archaeal motor assembly and motility from structures, conformations, and genetics. Molecular Cell. 49:1069-1082, 2013.
5. Appel MJ, Meier KK, Lafrance-Vanasse J, Lim H, Tsai CL, Hedman B, Hodgson KO, Tainer JA, Solomon EI, Bertozzi CR. Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation. Proc Natl Acad Sci USA. 116:5370-5375, 2019.
6. Tsai CL, Tripp P, Sivabalasarma S, Zhang C, Rodriguez-Franco M, Wipfler RL, Chaudhury P, Banerjee A, Beeby M, Whitaker RJ, *Tainer JA, Albers SV. The structure of the periplasmic FlaG-FlaF complex and its essential role for archaellar swimming motility. Nature Microbiol. 5:216-225, 2020 *co-corresponding author.
7. Hou J, Zhao R, Xia W, Chang CW, You Y, Hsu JM, Nie L, Chen Y, Wang YC, Liu C, Wang WJ, Wu Y, Ke B, Hsu JL, Huang K, Ye Z, Yang Y, Xia X, Li Y, Li CW, Shao B, Tainer JA, Hung MC. PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis. Nat Cell Biol. 22: 1264-1275, 2020.
8. Brosey CA, Houl JH, Katsonis P, Balapiti-Modarage LPF, Bommagani S, Arvai A, Moiani D, Bacolla A, Link T, Warden LS, Lichtarge O, Jones DE, Ahmed Z, Tainer JA. Targeting SARS-CoV-2 Nsp3 macrodomain structure with insights from human poly(ADP-ribose) glycohydrolase (PARG) structures with inhibitors. Prog Biophys Mol Biol. S0079-6107(21)00007-9, 2021.
9. Ye Z, Shi Y, Lees-Miller SP, Tainer JA. Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy. Front Immunol. 12:797880, 2021
10. Yuan X, Duan Y, Xiao Y, Sun K, Qi Y, Zhang Y, Ahmed Z, Moiani D, Yao J, Li H, Zhang L, Yuzhalin AE, Li P, Zhang C, Badu-Nkansah A, Saito Y, Liu X, Kuo WL, Ying H, Sun SC, Chang JC, Tainer JA, Yu D. Vitamin E Enhances Cancer Immunotherapy by Reinvigorating Dendritic Cells via Targeting Checkpoint SHP1. Cancer Discov. 12: 1742-1759, 2022.

Inhibitors provide incredibly powerful tools to probe and advance biology. By developing specific inhibitors for Mre11 exonuclease and endonuclease activities, for example, we discovered that homologous recombination is initiated by an Mre11 endonuclease nick, which in effect licenses HDR. Subsequent Mre11 exonuclease excision helps to create ssDNA as the committed step for HDR. Moreover, our DNA repair inhibitors can allow control of pathway choice at DNA double-strand breaks and replication forks. We envision these tools reverse the phenotype from cancer-causing defects in BRAC2 by redirecting repair to NHEJ instead of homologous recombination. Building upon these concepts, we are defining the roles of the GRB2 adaptor protein in HDR for both DSBs and for replication fork stress. We showed how GRB2 targets MRE11 nuclease for HDR. Furthermore, we find that nuclear GRAB2 (nGRAB2) is a biomarker for breast cancer progression and HDR proficiency.

Motivated by ongoing clinical trials with ATR inhibitors, we searched and identified the structure-specific nuclease EXO5 that is phosphorylated by ATR in response to replication fork stress. We found that ATR phosphorylation of EXO5 was critical for ATR-dependent replication fork restart. In fact, high EXO5 expression is linked to poor patient prognosis suggesting EXO5 may be a superior inhibitor target for oncogenic replication stress to ATR. Our EXO5 structures with and without DNA substrates suggest a target site for our door-stopped inhibitor strategy.

The success of PARP1 inhibitors in the clinic coupled to the unfortunate development of therapeutic resistance has promoted us to examine and seek to defeat PARP1 inhibitor resistance mechanisms. In this effort, we have solved structures and developed inhibitors for the Poly(ADP-ribose) glycohydrolase (PARG) that acts to remove PARylation. These PARGi control responses to DNA breaks and stalled forks. Moreover, they can overcome PARP1 inhibitor resistance and specifically kill cancer cells, likely due to their high levels of oncogenic replication stress.

Due to the success of cis-platinum and alkylating agents for cancer therapies and the observation that nucleotide excision repair (NER) can act in therapeutic resistance, we have solved X-ray crystal and cryo-EM structures of NER enzymes and will  employ our structures to design inhibitors targeting the XPD helicase and the NER nucleases. An active project is targeting the ERCC1-XPA interface with inhibitor tools that have promise as potential cancer therapeutics.

The Tainer Laboratory has developed a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis, as exemplified by specific targeting of (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with huge numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins.

As an overview of our methodology, we begin by harnessing Evolutionary Trace (ET) methods to distinguish conserved and variable sequence areas of the active site pocket. Based upon an evolutionary perspective of surface sequence conservation, we focus on sites offering optimal specificity. Next, we use our CASSIE approach of in silico screening of our selective GLAD library against available protein structures and experimental validation by binding or SAXS assays. By employing a chemically diverse and tractably sized GL library of compounds larger than fragments but smaller than drugs, we collapse potentially immense chemical library space into a sparse set of cell-friendly chemotypes. Our tactical approach for efficient computational screens is to identify the top 100 candidates for binding measurements. Top binding candidates are employed for crystallization experiments to obtain X-ray crystal structures of inhibitor candidates with micromolar or better binding. When human structures are not available, we developed a method for useful crystallographic feedback by employing avatars that incorporate essential target site features. We use early structural results to provide a rational basis to build focused chemical libraries to improve binding affinity plus favorable ADME properties and toxicological chemotypes. Together our GLAD library and CASSIE pipeline can efficiently provide inhibitor tools for cell biology and leads for preclinical drug discovery.

Exemplary Publications:

1. Daniels DS, Mol CD, Arvai AS, Kanugula S, Pegg AE, Tainer JA. Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. EMBO J. 19:1719-1730, 2000.
2. Putnam, C.D., Arvai, A.S., Bourne, Y., and Tainer, J.A. Active and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism. Journal of Molecular Biology. 296:295-309, 2000.
3. Doi Y, Katafuchi A, Fujiwara Y, Hitomi K, Tainer JA, Ide H, Iwai S. Synthesis and characterization of oligonucleotides containing 2'-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction. Nucleic Acids Res. 34:1540-51, 2006.
4. Garcin ED, Arvai AS, Rosenfeld RJ, Kroeger MD, Crane BR, Andersson G, Andrews G, Hamley PJ, Mallinder PR, Nicholls DJ, St-Gallay SA, Tinker AC, Gensmantel NP, Mete A, Cheshire DR, Connolly S, Stuehr DJ, Aberg A, Wallace AV, Tainer JA, Getzoff ED. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat Chem Biol. 4:700-7, 2008.
5. Perry JJ, Harris RM, Moiani D, Olson AJ, Tainer JA. J p38alpha MAP kinase C-terminal domain binding pocket characterized by crystallographic and computational analyses. Mol Biol. 391:1-11, 2009.
6. Omanakuttan A, Nambiar J, Harris RM, Bose C, Pandurangan N, Varghese RK, Kumar GB, Tainer JA, Banerji A, Perry JJ, Nair BG. Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Mol Pharmacol. 82:614-22, 2012.
7. Moiani D, Ronato DA, Brosey CA, Arvai AS, Syed A, Masson JY, Petricci E, Tainer JA. Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities. Methods Enzymol. 601:205-241, 2018.
8. Houl JH, Ye Z, Brosey CA, Balapiti-Modarage LPF, Namjoshi S, Bacolla A, Laverty D, Walker BL, Pourfarjam Y, Warden LS, Babu Chinnam N, Moiani D, Stegeman RA, Chen MK, Hung MC, Nagel ZD, Ellenberger T, Kim IK, Jones DE, Ahmed Z, and Tainer JA. Selective small molecule PARG inhibitor causes replication fork stalling and cancer cell death. Nat Commun. 10:5654, 2019.
9. Tainer JA, Ahmed Z, Ye Z.  Methods For Treating Cancers With Inhibitors Targeting The Role of GRB2 In DNA Repair. Patent UTSC.P1507US.P1, 2020.
10. Zhou J, Gelot C, Pantelidou C, Li A, Yücel H, Davis RE, Farkkila A, Kochupurakkal B, Syed A, Shapiro GI, Tainer JA, Blagg BSJ, Ceccaldi R, D'Andrea AD. A first-in-class Polymerase Theta Inhibitor selectively targets Homologous-Recombination-Deficient Tumors. Nat Cancer. 2:598-610, 2021.
11. Moiani D, Link TM, Brosey CA, Katsonis P, Lichtarge O, Kim Y, Joachimiak A, Ma Z, Kim IK, Ahmed Z, Jones DE, Tsutakawa SE, Tainer JA. An efficient chemical screening method for structure-based inhibitors to nucleic acid enzymes targeting the DNA repair-replication interface and SARS CoV-2. Methods Enzymol. 661:407-431, 2021.
12. Tainer JA, Ahmed Z, Jones DE, Kim I, Ellenberger T, Ho C. Small molecule PARG inhibitors and methods of use thereof. Patent US 17600049, 2022.

The Tainer Laboratory has successfully trained scores of postdoctoral fellows, graduate students and undergraduates, most of whom have gone on to successful careers in science and research. We maintain a positive, open and interactive research environment that attracts talented scientists from around the world. We believe in the value of developing advanced “platforms,” i.e., systems of enabling technologies and associated institutional and human infrastructure to provide firm foundations for breakthroughs.

Our training plan recognizes the needs to develop top talent, to preserve open environments that foster disruptive discoveries and to take full advantage of the rich Houston research environment in terms of people and technologies. Participation in our interactive, transdisciplinary teams of leading clinical and scientific researchers at MD Anderson plus our international NCI Program collaborators helps our trainees learn to effectively design experiments and link basic and biomedical research. The Tainer Lab aims to teach students and postdocs all the skills needed to run a research laboratory including writing proposals, publishing important papers and responding to reviewers. Our trainees learn to combine biochemistry and molecular biology with new protein structures and biophysics and with cellular phenotypes. They gain experience optimizing target protein production plus a spectrum of biophysical methods along with conceptual and applied expertise in structural biology including X-ray crystallography, small angle X-ray scattering and cryo-electron microscopy. The Tainer Lab teaches trainees how to analyze and learn from structures and to test implications with biochemistry, mutations and cell biology. Group members learn how to write impactful papers on structure and mechanism that inform cell biology, complete their dissertations, write fundable proposals, work effectively with collaborators and respond to reviewer criticism. These skills enable advanced research investigating how structure and mechanism can drive biological outcomes including both physiological and pathological events.

This is a magical time in biological research as advances in both equipment and technologies have made experiments extremely powerful and efficient. Timely results can be obtained for new biology and biomedical research that can impact how others do their experiments as well as advance clinical efforts. In this context, MD Anderson Cancer Center plus the university and medical center environment of Houston is a uniquely empowering combination for both advanced training and breakthrough research accomplishments.