James L. Keck
Position title: Professor; also Associate Dean for Basic Sciences, UW SMPH
Phone: (608) 263-1815
6214A Biochemical Sciences Building
440 Henry Mall, Madison, WI 53706
• B.S. 1992, University of Massachusetts
• Ph.D. 1997, University of California-Berkeley (S. Marqusee)
• Postdoctoral, 1997-98, Harvard University (J. Wang);1999-01, University of California-Berkeley (J. Berger)
Honors & Awards
• Jane Coffin Childs Memorial Fund Postdoctoral Fellow, 1997-2000
• Shaw Scientist Award, 2003
• American Cancer Society Research Scholar, 2006
• WARF Romnes Faculty Fellowship, 2009
• School of Medicine and Public Health Dean’s Teaching Award, 2010
• UW Health Community Service Award 2013
• UW Madison Kellett Mid-Career Award, 2017
• Medical Alumni Association Distinguished Basic Science Teaching Award, 2017
Research in the Keck lab examines the structural mechanisms that drive DNA replication, replication restart, recombination, and repair reactions. Successful execution of these pathways is essential in all cells, and defects in the proteins that facilitate genome maintenance reactions lead to genome instability, cell death, and disease. Our studies combine structural approaches with biochemical and cell biological methods to answer fundamental structure-function questions in genome biology.
Single-stranded DNA-binding proteins. Bacterial single-stranded (ss) DNA-binding proteins (SSBs) play essential protective and organizational roles in genome biology. In their protective functions, SSBs bind and sequester ssDNA intermediates that are formed during genome maintenance reactions. As organizational centers, SSB/ssDNA complexes form dynamic protein-docking “hubs” at which over a dozen different DNA replication, recombination, and repair enzymes gain access to genomic substrates through direct interactions with SSB. This clustering of enzymes is thought to help integrate cellular genome maintenance reactions by facilitating the exchange of ssDNA substrates between DNA replication, recombination, and repair pathways. In all cases examined to date, the last ~6 residues of SSB’s flexible C-terminus (SSB-Ct) form its protein docking site. Eukaryotic SSBs also interact with a diverse array of genome maintenance proteins but, since they lack the SSB-Ct element found in bacterial SSBs, they do so through distinct mechanisms.
We have mapped the SSB-Ct binding sites in several genome maintenance proteins. The examples we have focused on include bacterial proteins involved in DNA replication (chi subunit from the replicative DNA polymerase), recombination (RecQ DNA helicase), and repair (Exonuclease I and Uracil DNA glycosylase). The SSB-docking sites on these proteins are remarkably similar in spite their lack of structural similarity. Binding to SSB often stimulates enzyme activity through recruitment of the protein partner to its SSB/DNA substrate. In cells, these interactions are essential for the proper localization and functions of SSB-associated proteins.
Future work aims to establish the full extent of the SSB interaction network though protein interaction studies. We plan to examine how multiple SSB-binding proteins might function cooperatively on SSB/ssDNA substrates and to determine the mechanisms that regulate assembly of SSB-interacting proteins on SSB/ssDNA complexes in cells. The essential nature of bacterial SSB/protein interactions also makes the complexes attractive targets for the development of novel antibacterial therapeutics that block SSB/protein interactions.
Mechanisms of DNA replication restart. Nearly all replication complexes (replisomes) formed at bacterial replication origins are thought to stall during the course of DNA replication. This stalling may occur at sites of DNA damage and can lead to dramatic rearrangement of the replication fork DNA and disassembly of the replisome. Reassembly of the DNA replication machinery on abandoned replication forks is therefore often required for complete DNA replication. “DNA replication restart” is mediated several proteins (PriA, PriB, PriC, and DnaT in E. coli) that function in a coordinated manner to reload the replisome. Currently, little is known about the structural mechanisms that support this essential cellular function.
We have developed a model based on work from our group and others that explains the linkage between abandoned replication fork recognition and replisome reloading. In this model, PriA acts as a first-responder protein, binding directly to the abandoned replication fork DNA. Subsequent stepwise assembly of PriB and DnaT or PriC onto the PriA/DNA complex creates a platform for recruiting the replisome on the replication fork. As a step toward understanding the structure of the primosome, we have determined the structures of PriA and PriB and have initiated structural efforts on the remaining replication restart proteins. Our future work will focus on understanding the critical steps in formation of the fully assembled primosome through crystallographic and other structural approaches, along with biochemical and genetic studies.
Coordination of human DNA repair complexes. Eukaryotic cells have an astonishing number of DNA repair pathways that are regulated and coordinated, at least in part, by dynamic physical interactions. We have recently begun a study of a key protein interaction interface that links the Fanconi Anemia Core Complex and Bloom Dissolvasome, two DNA repair complexes. Our lab has determined the high-resolution crystal structure of the minimal protein interface that links these complexes, which is comprised of a short peptide from the FANCM protein from the Fanconi Anemia Core Complex binding to the RMI heterodimer from the Bloom Dissolvasome. Using our structure as a guide, we have shown that mutations that block interaction between these complexes induces genomic instability in cells-a hallmark of nearly all cancers. We are continuing to investigate this and other repair interfaces as examples of regulation by dynamic protein assembly and as potential chemotherapeutic targets.
Publications of Note
Perform a customized PubMed literature for Dr. Keck.
• Windgassen, TA, Leroux, M, Satyshur, KA, Sandler, SJ & Keck, JL (2018) “Structure-specific DNA replication fork recognition directs helicase and replication restart activities of the PriA DNA helicase” Proceedings of the National Academy of Sciences (in press).
• Bagchi, D, Manosas, M, Zhang, W, Manthei, KA, Hodeib, S, Ducos, B, Keck, JL, Croquette, V (2018) “Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB” Nucleic Acids Res (in press).
• Voter, AF, Keck, JL (2018) “Development of protein-protein interaction inhibitors for the treatment of infectious diseases” Adv Protein Chem Struct Biol 111, 197-222.
• Windgassen, TA, Wessel, SR, Bhattacharyya, B, Keck, JL (2018) “Mechanisms of bacterial DNA replication restart” Nucleic Acids Research 46, 504-519.
• Voter, AF, Killoran, MP, Anavive, GE, Wildman, S, Hoffmann, FM & Keck, JL (2018) “A high-throughput screening strategy to identify inhibitors of SSB protein-protein interactions in an academic screening facility” SLAS Discovery 1, 94-101.
• Windgassen, TA & Keck, JL (2016) “An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase” Nucleic Acids Research 44, 9745-9757.
• Wessel, SR, Cornilescu, CC, Cornilescu, G, Sandler, SJ, Markley, JL & Keck, JL (2016) “Structure and function of the PriC DNA replication restart protein” Journal of Biological Chemistry 291, 18384-18396.
• Voter, AF, Manthei, KA & Keck, JL (2016) “A high throughput screening strategy to identify protein-protein interaction inhibitors that block the Fanconi anemia DNA repair pathway” Journal of Biomolecular Screening 21, 626-633.
• Petzold, C, Marceau, AH, Miller, KH, Marqusee, S & Keck, JL (2015) “Interaction with single-stranded DNA-binding protein stimulates Escherichia coli ribonuclease HI enzymatic activity” Journal of Biological Chemistry 290, 14626-14636.
• Manthei, KA, Hill, MC, Burke, JE, Butcher, SE & Keck, JL (2015) “Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases” Proceedings of the National Academy of Sciences 114, 4292-4297.
• Liu, K, Krasny, K, Myers, AR, Satyshur, KA, Keck, JL & Wang, JD (2015) “Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp” Molecular Cell 57, 735-749.