Professional Education

  • Bachelor of Science, Bradley University (2011)
  • Doctor of Philosophy, University of Wisconsin Madison (2018)

All Publications

  • Function of a strand-separation pin element in the PriA DNA replication restart helicase JOURNAL OF BIOLOGICAL CHEMISTRY Windgassen, T. A., Leroux, M., Sandler, S. J., Keck, J. L. 2019; 294 (8): 2801–14
  • The elemental mechanism of transcriptional pausing ELIFE Saba, J., Chua, X., Mishanina, T. V., Nayak, D., Windgassen, T. A., Mooney, R., Landick, R. 2019; 8


    Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

    View details for PubMedID 30618376

  • Function of a strand-separation pin element in the PriA DNA replication restart helicase. The Journal of biological chemistry Windgassen, T. A., Leroux, M., Sandler, S. J., Keck, J. L. 2018


    DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA crosslinking, we localized the putative pin (a beta-hairpin within a zinc-binding domain in PriA) near the ss/ds DNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the beta-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA beta-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted beta-hairpin PriA variant displayed wild type levels of DNA unwinding and PriB binding in vitro. These results suggest essential but sequence non-specific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.

    View details for PubMedID 30593500

  • Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Windgassen, T. A., Leroux, M., Satyshur, K. A., Sandler, S. J., Keck, J. L. 2018; 115 (39): E9075–E9084


    DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA's structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.

    View details for PubMedID 30201718

  • Mechanisms of bacterial DNA replication restart NUCLEIC ACIDS RESEARCH Windgassen, T. A., Wessel, S. R., Bhattacharyya, B., Keck, J. L. 2018; 46 (2): 504–19


    Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved 'DNA replication restart' pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT).

    View details for PubMedID 29202195

    View details for PubMedCentralID PMC5778457

  • Integrase Move-In Day: Mechanisms of HIV DNA Integration Windgassen, T. A., Fahlberg, S., Hanson, B., Klauser, K., Semia, S., Keck, J. L. FEDERATION AMER SOC EXP BIOL. 2017
  • An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase NUCLEIC ACIDS RESEARCH Windgassen, T. A., Keck, J. L. 2016; 44 (20): 9745–57


    Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue- and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms.

    View details for PubMedID 27484483

    View details for PubMedCentralID PMC5175346

  • Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation DNA REPAIR Piechura, J. R., Tseng, T., Hsu, H., Byrne, R. T., Windgassen, T. A., Chitteni-Pattu, S., Battista, J. R., Li, H., Cox, M. M. 2015; 26: 30–43


    Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism.

    View details for PubMedID 25559557

    View details for PubMedCentralID PMC4308431

  • Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase NUCLEIC ACIDS RESEARCH Windgassen, T. A., Mooney, R., Nayak, D., Palangat, M., Zhang, J., Landick, R. 2014; 42 (20): 12707–21


    The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH-NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.

    View details for PubMedID 25336618

    View details for PubMedCentralID PMC4227799

  • RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement NATURE STRUCTURAL & MOLECULAR BIOLOGY Hein, P. P., Kolb, K. E., Windgassen, T., Bellecourt, M. J., Darst, S. A., Mooney, R. A., Landick, R. 2014; 21 (9): 794–802


    The rates of RNA synthesis and the folding of nascent RNA into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA-exit channel can either increase pausing by interacting with RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain has been proposed to mediate some effects of nascent-RNA structures. However, the connections among RNA structure formation and RNAP clamp movement and catalytic activity remain uncertain. Here, we assayed exit-channel structure formation in Escherichia coli RNAP with disulfide cross-links that favor closed- or open-clamp conformations and found that clamp position directly influences RNA structure formation and RNAP catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening through interactions with the flap that slow translocation.

    View details for PubMedID 25108353

    View details for PubMedCentralID PMC4156911

  • A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science Larson, M. H., Mooney, R. A., Peters, J. M., Windgassen, T., Nayak, D., Gross, C. A., Block, S. M., Greenleaf, W. J., Landick, R., Weissman, J. S. 2014; 344 (6187): 1042-1047


    Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.

    View details for DOI 10.1126/science.1251871

    View details for PubMedID 24789973

  • Cys-Pair Reporters Detect a Constrained Trigger Loop in a Paused RNA Polymerase MOLECULAR CELL Nayak, D., Voss, M., Windgassen, T., Mooney, R., Landick, R. 2013; 50 (6): 882–93


    Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.

    View details for PubMedID 23769674

    View details for PubMedCentralID PMC4037917