Investigation of Intradomain Motions of a Y-Family DNA Polymerase during Substrate Binding and Catalysis

Abstract

DNA polymerases catalyze DNA synthesis through a stepwise kinetic mechanism that begins with binding to DNA, followed by selection, binding, and incorporation of a nucleotide into an elongating primer. It is hypothesized that subtle active site adjustments in a polymerase to align reactive moieties limit the rate of correct nucleotide incorporation. DNA damage can impede this process for many DNA polymerases, causing replication fork stalling, genetic mutations, and potentially cell death. However, specialized Y-family DNA polymerases are structurally evolved to efficiently bypass DNA damage in vivo, albeit at the expense of replication fidelity. Dpo4, a model Y-family polymerase from Sulfolobus solfataricus, has been well-studied kinetically, structurally, and computationally, which yielded a mechanistic understanding of how the Y-family DNA polymerases achieve their unique catalytic properties. We previously employed a real-time Förster resonance energy transfer (FRET) technique to characterize the global conformational motions of Dpo4 during DNA binding as well as nucleotide binding and incorporation by monitoring changes in distance between sites on the polymerase and DNA, and even between domains of Dpo4. Here, we extend the utility of our FRET methodology to observe conformational transitions within individual domains of Dpo4 during DNA binding and nucleotide incorporation. The results of this novel, intradomain FRET approach unify findings from many studies to fully clarify the complex DNA binding mechanism of Dpo4. Furthermore, intradomain motions in the Finger domain during nucleotide binding and incorporation, for the first time, report on the rate-limiting step of a single-nucleotide addition catalyzed by Dpo4.

Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

“…The intermediate state caused by stable individual domain folding during DPO4 folding from the default SBM simulations resonates with the experimentally observed unfolding intermediate ( Sherrer et al, 2012 ). In addition, simulations with the default SBM have described a multistate process for DPO4–DNA binding, in good agreement with the results of previous experiments, where the complexity of the processes, including multistep DNA binding and multistate DPO4 conformational dynamics, was revealed ( Maxwell et al, 2014 ; Raper and Suo, 2016 ; Lee et al, 2017 ). These features are manifestations that the native topologies of DPO4 and the DPO4–DNA complex are major elements in determining the mechanisms of folding ( Clementi et al, 2000 ) and binding ( Levy et al, 2004 ), respectively.…”

“…We expect to see a more cooperative folding of DPO4 with more synchronous melting curves of different DPO4 truncation mutants through weakening the intradomain interactions. In addition, the previously designed stopped-flow and single-molecule FRET experiments revealed a dynamical conformational equilibrium of DPO4 during DNA binding ( Xu et al, 2009 ; Maxwell et al, 2012 ; Raper and Suo, 2016 ; Raper et al, 2016 ). These FRET-based techniques have further measured the kinetic rates of DPO4 interconversion between different states during DNA binding ( Raper et al, 2018 ).…”

“…Thermal denaturation experiments revealed that unfolding of truncated DPO4 mutants, such as the DPO4 core and LF domains, proceeds via cooperative processes ( Sherrer et al, 2009 ). Stopped-flow Förster resonance energy transfer (FRET) studies monitoring intradomain ( Raper and Suo, 2016 ) and interdomain ( Xu et al, 2009 ; Maxwell et al, 2014 ) conformational dynamics of DPO4 during DNA binding as well as nucleotide binding and incorporation revealed weak and strong interactions between and within each domain of DPO4, respectively. Using only topological information, an SBM with homogeneously weighted native contacts predicted a ‘divide-and-conquer’ domain-wise folding scenario for DPO4 folding ( Wang et al, 2012a ; Chu et al, 2020 ).…”

“…It is worth noting that we did not use this ‘double-basin’ SBM for investigating DPO4 folding, where the potential of the SBM ( ) has only a ‘single basin’ in the apo-DPO4 structure. This is because (1) in the absence of DNA, DPO4 is mostly in apo form, and the transition rate for DPO4 from apo form to DNA binary form is very slow ( Raper and Suo, 2016 ; Lee et al, 2017 ), so DPO4 is prone to fold to its apo form without DNA, and (2) the contacts formed at the F–LF interface can be regarded as a consequence of DNA binding ( Raper and Suo, 2016 ), so the formation of these contacts reflects the effect of DNA binding. On the other hand, DNA was kept frozen throughout the simulations.…”

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants

“…Based on previous mutational studies demonstrating an increase in processivity in polymerases, telomerase, and TERT, we hypothesized that the processivity of Dpo4 would be similarly affected. Dpo4 shares a similar conformation with the replicative polymerase, that is, a conserved right-hand structure composed of palm, finger, and thumb domains, plus a unique "little finger" (LF) domain at the C terminus (6,23). In the interaction between Dpo4 and DNA, the thumb domain contacts the backbone of the primer and the template strands through the minor groove.…”