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. In collaboration with Joe Loparo we reconstituted TLS at the single-molecule level and showed that the E. coli b clamp can simultaneously bind the replicative polymerase Pol III and DinB enabling exchange of the two polymerases and rapid bypass of a DinB cognate lesion. 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-guanosine into newly synthesized RNAs. Additional support for this model comes from our recent observation that overproduction of MutS suppresses ampicillin lethality.
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 non-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 in collaboration with Dmitry Korzhnev and Pei Zhou 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 in collaboration with Pei Zhou 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 highly conserved bacterial RNase, YbeY, that plays crucial roles in in 70S ribosome quality control, rRNA processing, and small RNA regulation. Recent interests include investigations of how the rhizobial cell cycle is controlled both by cellular factors and by plant-encoded NCR peptides that force the terminal differentiation of the rhizobia into bacteroids. By developing the first-ever procedure for robustly synchronizing S. meliloti we were able to carry out a detailed analysis of striking changes in gene expression during the S. meliloti cell cycle, thereby enabling a comparison with the intensively studied Caulobacter crescentus system. We showed that sub-lethal levels of one of these plant peptides, NCR247, prevented Z-ring formation, robustly blocked cell division in G2 phase, and significantly altered the expression of about 15% of total genes.
Teaching and Education
I have been deeply involved in teaching and undergraduate education throughout my career. I was in charge of the MIT undergraduate program in Biology for a total of 22 years, have directed MIT’s HHMI-funded program in undergraduate education in the biological sciences since its inception in 1989, and 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. I 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.
Cohen, S.E, C.A. Lewis, R.A. Mooney, M.A. Kohanski, J.J. Collins, R. Landick and 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).
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).
Foti, J.J., and G.C. Walker. Efficient Extension of Slipped DNA Intermediates by DinB (Pol IV) is Required to Escape Primer Template Realignment by DnaQ. J. Bacteriol. 193:2637-2641 (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).
Pini, F., B. Frage, L. Ferri, N.J. De Nisco, S.S. Mohapatra, L. Taddei, A. Fioravanti, F. Dewitte, M. Galardini, M. Brilli, V. Villeret, M. Bazzicalupo, A. Mengoni, G.C. Walker, A. Becker. and E. G. Biondi. The essential DivJ/CbrA kinase and PleC phosphatase system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti. Mol. Microbiol. 90:54-71 (2013).
Xu, X., K. Xie, X.-Q. Zhang, E. Pridgen, G. Y. Park, D. Cui, J. Shi, S. Lippard,, G. Walker, and O. Farokhzad. Enhancing Tumor Cells Response to Chemotherapy through Nanoparticle-Mediated Co-delivery of siRNA and Cisplatin Prodrug. Proc. Natl. Acad. Sci. U.SA. 110:18638-18643 (2013).
De Nisco, N.J., R.P. Abo, C.M. Wu, J. Penterman, and G.C. Walker. Global Analysis of Cell Cycle Gene Expression of the Legume Symbiont Sinorhizobium meliloti. Proc. Natl. Acad. Sci. U.SA. 111:3217-3224 (2014).
Penterman, J., R.P. Abo, N.J. De Nisco, M.F. F. Arnold, R. Longhi, M. Zanda, and G. C. Walker. Host Plant Peptides Elicit a Transcriptional Response to Control the Sinorhizobium meliloti Cell Cycle During Symbiosis. Proc. Natl. Acad. Sci. U.SA. 111:3561-3566 (2014).
Pandey, S.P., J.A. Winkler, H. Li, D. M. Camacho, J.J. Collins, and G.C. Walker. Central Role for RNase YbeY in Hfq-Dependent and Hfq-Independent Small-RNA Regulation in Bacteria. BMC Genomics 15:121 (2014).
Dwyer, D.J., P. Belenky, J.H. Yang, I.C. MacDonald, J.D. Martell, N. Takahashi, C.T.Y. Chan, M.A. Lobritz, D. Braff, E.G. Schwarz, J.D. Ye, M. Pati, M. Vercruysse, P.S. Ralifo, K.R. Allison, A.S. Khalil, A.Y. Ting, G.C. Walker, and J.J. Collins. Antibiotics Induce Redox-related Physiological Alterations as Part of their Lethality. Proc. Natl. Acad. Sci. U.SA. 111:2100-2109 (2014).
Kath, J.E., S. Jergic, J.M.H. Heltzel, D.T. Jacob, N.E. Dixon, M.D. Sutton, G.C. Walker, and J.J. Loparo. Polymerase Exchange on Single DNA Molecules Reveals Processivity Clamp Control of Translesion Synthesis. Proc. Natl. Acad. Sci. U.S.A. 111:7647-52 (2014).
Vercruysse, C. Köhrer, B.W. Davies, M.F.F. Arnold, J.J. Mekalanos, U.L. RajBhandary, G.C. Walker. The Highly Conserved Bacterial RNase YbeY is Essential in Vibrio cholerae, Playing a Critical Role in Virulence, Stress Regulation, and RNA Processing. PLoS Pathogens. 10:e1004 (2014).
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. (2006).