Our research is concentrated in two major areas. The first concerns the regulation and mechanism of action of proteins involved in DNA repair and mutagenesis and in other cellular responses to DNA damage. The second deals with identification of the bacterial functions required for the development of nitrogen-fixing nodules on legumes and with the relationship between rhizobial functions required for nodule invasion/infection and mammalian pathogenesis. Both lines of research have also offered unexpected insights into other important areas of biology
DNA repair and mutagenesis in bacteria
Translesion DNA synthesis (TLS) carried out by specialized DNA polymerases is an important mechanism of DNA damage tolerance in all domains of life. In bacteria, most mutations that result from DNA damage are the consequence of umuDC-dependent mutagenic TLS. Cleavage of UmuD in a RecA-mediated fashion plays a key role in controlling the function of the UmuC. In addition to UmuD’2C (DNA pol V), Escherichia coli encodes a second Y family DNA polymerase, DinB (DNA pol IV). Our discovery that DinB is a much better polymerase when copying over N2-furfuryl-dG than over ordinary dG suggests a biological role of carrying out relatively accurate DNA synthesis over a common class of N2-dG adducts, while the ‑1 frameshift mutator activity of modestly overproduced DinB can be explained by template slippage. Our efforts to understand why DinB overproduction is cytotoxic to E. coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We have proposed a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.
DNA repair and mutagenesis in eukaryotes
In, eukaryotes, most mutations that result from DNA damage are the consequence of REV1/3/7-dependent mutagenic TLS. Rev1 is a Y family DNA polymerase and pol ζ (Rev3/Rev7) is a B family DNA polymerase. In collaboration with Mike Hemann,we have previously shown that knocking down REV1 expression in the Eμ-myc mouse lymphoma model strikingly reduces acquired drug resistance when tumors relapse, while knocking down REV3 in a mouse model of human small cell lung cancer made these essentially untreatable tumors responsive to DNA damaging chemotherapy. Additional evidence that interfering with REV1/3/7-dependent mutagenic TLS might have the potential to improve chemotherapy is provided by our demonstration that nanoparticles carrying both siRNAs directed against REV1 and REV3 and cisplatin as pro-drug completely suppress tumor growth in a prostate xenograft model. Rev1 has only a limited ability to insert nucleotides opposite a lesion, but has a C-terminal domain (CTD) that plays a critical role in TLS by recruiting other TLS DNA polymerases. We had previously shown that the Rev1 CTD interacts with the Rev7 subunit of DNA pol ζ and had predicted that it is composed of four amphipathic helixes. NMR studies revealed that the mammalian Rev1 CTD is an atypical four-helix bundle with one interaction site for Rev7 and another for DNA pols κ, η, and ι. The subsequent structural determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ Rev1-interacting region is helping to guide our efforts to develop novel cancer therapeutics that suppress TLS.
The Sinorhizobium-legume symbiosis
We also study the symbiosis between legumes and the nitrogen-fixing bacterium Sinorhizobium meliloti. Over the years, our studies have revealed the symbiotic roles of the succinoglycan and EPS II exopolysaccharides, lipopolysaccharides, and the BacA protein among others. This line of research has also led to unexpected findings including the discovery of the “missing step” in vitamin B12 biosynthesis, insights into commonalities between the Rhizobium-legume symbiosis and the chronic intracellular infections caused by the human pathogen Brucella, and the discovery of new RNase, YbeY, that plays crucial roles in in 70S ribosome quality control, rRNA processing, and small RNA regulation. Recent interests include investigation of how the rhizobial cell cycle is controlled both by cellular factors and by plant-encoded NCR peptides.
Teaching and Education
I have been deeply involved in teaching and undergraduate education throughout my career. For a total of 20 years, I has been in charge of the MIT undergraduate program in Biology, have directed MIT’s HHMI-funded program in undergraduate education in the biological sciences since its inception in 1989, and have served as the Housemaster of McCormick Hall. in 2002, and again in 2010, I was awarded a four-year HHMI Professorship to support my efforts in undergraduate education. He used those funds to establish an Education Group, whose accomplishments included the development of StarBiochem, an internationally used, freely available 3D protein viewer designed for education, StarGenetics, a Mendelian cross simulator, and the ongoing development of StarCellBio, a simulator of cell and molecular biology experiments. Current projects including assisting with the development of MITx/edX courses.
Foti, J.J., A.M. DeLucia, C.M. Joyce, and G.C. Walker. A Non-Covalent Step In the Escherichia coli DinB Template Slippage Pathway Is Inhibited By UmuD2. J. Biol. Chem. 285:23086-95 (2010).
Cohen, S.E, C.A. Lewis, R.A. Mooney, M.A. Kohanski, J.J. Collins, R. Landickand G.C. Walker. Roles for the Transcription Elongation Factor NusA in Both DNA Repair and Damage Tolerance Pathways in Escherichia coli. Proc. Natl. Acad. Sci. USA. 107:15517-15522 (2010).
Bomar, M.G., S. D'Souza,, M. Bienko, I. Dikic, G.C Walker, and P. Zhou. Unconventional Ubiquitin Recognition by the Ubiquitin-Binding Motif within the Y-Family DNA Polymerases iota and Rev1. Mol. Cell 37:408-417 (2010).
Barra-Bily, L., S.P. Pandey, A. Trautwetter, C. Blanco, and G.C. Walker. The Sinorhizobium meliloti RNA chaperone Hfq Mediates Symbiosis of S. meliloti and alfalfa. J. Bacteriol.192:1710-1718 (2010).
Taga, M.E. and G.C. Walker. Sinorhizobium meliloti Requires a Cobalamin-Dependent Ribonucleotide Reductase for Symbiosis with its Plant Host. Mol. Plant-Microbe Interact. 23:1643-1654 (2010).
Davies, B.W., C. Köhrer, A.I. Jacob, L.A. Simmons, J. Zhu, L.M. Aleman, U.L. RajBhandary, and G.C. Walker. Role of Escherichia coli YbeY, a Highly Conserved Protein, in rRNA Processing. Mol. Microbiol. 78:506-518 (2010).
Xie, K., J. Doles, M.T. Hemann, and G.C. Walker. Error-Prone Translesion Synthesis Mediates Acquired Chemoresistance. Proc. Natl. Acad. Sci. U.S.A. 107:20792-20797 (2010).
Doles, J., T.G Oliver, G. Hsu, T. Jacks, G.C. Walker, and M.T Hemann, Rev3 Suppression Sensitizes Drug Resistant Lung Tumors to Chemotherapy. Proc. Natl. Acad. Sci. U.S.A. 107:20786-20791 (2010).
Wiltrout, M.E. and G.C. Walker. The DNA Polymerase Activity of Saccharomyces cerevisiae Rev1 is Biologically Significant. Genetics. 187:21-35 (2011).
Pandey, S.P., B.K. Minesinger, and G.C. Walker. A Highly Conserved Protein of Unknown Function in Sinorhizobium meliloti Affects sRNA Regulation Similar To Hfq. Nucleic Acids Res. 39:4691-4708 (2011).
Modi, S.R., D.M. Camacho, M.A. Kohanski, G.C. Walker, and J.J. Collins. Functional Characterization of Bacterial sRNAs Using a Network Biology Approach. Proc. Natl. Acad. Sci U.SA. 108:15522-15527 (2011).
Foti, J.J., B. Devadoss, J. A. Winkler, J.J. Collins, and G.C.Walker. Oxidation of the Guanine Nucleotide Pool Underlies Cell Death by Bactericidal Antibiotics. Science. 336:315-319 (2012).
Pozhidaeva, A., Y. Pustovalova, S. D’Souza, I. Bezsonova, G.C. Walker, and D.M. Korzhnev. NMR Structure and Dynamics of the C-terminal Domain from Human Rev1 and its Complex with DNA Polymerase η. Biochemistry. 51:5506-5520 (2012).
Wojtaszek, J., C.-J. Lee, S. D'Souza, B. Minesinger, H. Kim, A.D. D'Andrea, G.C. Walker, and P. Zhou. Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric Pol z and Pol k. J. Biol. Chem. 287:33836-33846 (2012).
Jacob, A.I., C. Köhrer, B.W. Davies, U.L. RajBhandary, and G.C. Walker. Conserved Bacterial RNase YbeY Plays Key Roles in 70S Ribosome Quality Control and 16S rRNA Maturation. Mol. Cell. 49:427-38 (2013).
Friedberg, E.C., G.C. Walker, W. Siede, R.D. Wood, R.A. Schultz, T. Ellenberger. DNA Repair and Mutagenesis: Second Edition. American Society for Microbiology, Washington, D.C. (2005).