To determine whether the interaction of the excluded strand has effects on the unwinding ability of EcDnaB, several conserved surface-exposed mutations were created (Figure 2.10).
Electrostatic interactions were found to mediate the wrapping interaction in the case of SsoMCM, and titrating increasing amounts of salt onto the 30/30 WT EcDnaB complex resulted in increased dynamics (shorter dwell times), suggesting that EcDnaB also utilizes electrostatic interactions to mediate wrapping (Figure 2.11). Based on a homology model for EcDnaB, four surface mutants (R74A, R164A, K180A, and R328A/R329A) that exist in positively charged electrostatic patches (Figure 2.12A-B) were cloned, expressed, and purified. All mutants were consistent with forming a hexamer as the major peak after gel filtration.
Figure 2-10: Amino acid sequence alignments of DnaB helicases.
A) Multiple amino acid alignment of DnaB helicases using CLUSTAL W2 (http://www.ebi.ac.uk/Tools/clustalw2).
B) The phylogenetic tree was created using phyloT (http://phylot.biobyte.de). Identical (*), similar (:), and somewhat similar (.) residues are indicated. ECOLI - Escherichia coli strain (K-12); GEOSE - Geobacillus stearothermophilus; ECOBD - Escherichia coli strain (BL21-DE3); BARGA - Bartonella grahamii (strain as4aup) SALTY - Salmonella typhimurium; HELPY - Helicobacter pylori strain (26695); ECO57 - Escherichia coli
Figure 2-12: ExPRT plots of WT EcDnaB and selected mutants bound to different fork lengths.
Position of the mutations mapped onto the homology model for EcDnaB A) colored and B) electrostatic surfaces.
ExPRT plots comparing dwell times and probability of transition for C-E) WT and F) K180A, G) R74A, and H) R328A/R329A bound to 30/30, 40/30, and 50/30 substrates, respectively.
Figure 2-13: Example smFRET kinetic traces.
Comparison of A-C) WT and D) K180A, E) R74A, and F) R328A/R329A EcDnaB FRET efficiencies as a function of time on the 30/30, 40/30 and 50/30 forks, respectively. The calculated FRET values (blue) are overlaid with the
smFRET wrapping assays were performed for each mutant on each of the three fork templates and analyzed and compared using traditional histograms and newer ExPRT plots (Figure 2.14). A selection of ExPRT plots for various mutants are compared to WT EcDnaB on the same fork substrate (Figure 2.12). Several important differences between the hexamer-excluded strand interactions as well as the dynamics of those interactions are highlighted by the ExPRT plots. For example, EcDnaB (K180A) with the 30/30 fork produces similar FRET states and dynamics when compared to the wild-type EcDnaB; however, there are five states compared to three (Figure 2.12C & F). Example traces for individual molecules for wild-type compared to K180A are shown in Figure 2.13A & D. A greater number of FRET states and transitions are indicative of a less stable interaction between the exterior surface of the helicase and the excluded strand leading to alternative binding positions. Similarly, the R164A mutant also samples a greater number of states than wild-type, especially on the 40/30 and 50/30 substrates (Figure 2.14M & R). These results suggest that residues K180 and R164 contribute to but do not solely mediate the helicase-excluded strand interactions that give rise to the FRET states we observe for the wild-type.
Figure 2-14: Histograms and ExPRT Plots of WT and EcDnaB and mutants bound to DNA forks.
Histograms (A-E) report the population of molecules as a function of FRET states on DNA forks with a 30-base 5’-strand and a 30-base (blue), 40-base (green), or 50-base (red) 3’-5’-strand for WT, R74A, R164A, K180A, and R328A/R329A. Yellow, blue, and red regions highlight low, medium, and high FRET populations, respectively.
Corresponding ExPRT plots are shown for (F-J) 30/30, (K-O) 40/30, and (P-T) 50/30 forks for each of the respective EcDnaB helicases.
EcDnaB (R74A) on the longer fork substrates displays similar dynamics between two FRET states compared to WT (Figure 2.14G, L, Q vs. F, K, P). Wild-type EcDnaB yields more traces with transitions than R74A does, and R74A produces histograms and ExPRT plots that lack the high FRET state (~0.95) that we see in all other data sets (Figure 2.14). The absence of that highest FRET state for R74A across all substrates tested and the presence of that same highest FRET state for all other EcDnaB constructs with all three substrates suggests that R74 is necessary for that particular FRET state. R74 is positioned close to the top of the hexamer,
interaction at R74 would be consistent with the dyes being in extremely close proximity.
Therefore, the R74A mutant provides insight into the excluded strand binding path on the exterior surface that transverses the entire lateral length of the exterior surface.
The EcDnaB (R328A/R329A) mutant shows extreme differences in the binding and dynamics on the longer DNA strands compared to wild-type (Figure 2.14E, J, O, T). Wild-type EcDnaB on the 50/30 fork shows a small fraction of traces that transition between high and medium FRET states (Figure 2.12E). In comparison, EcDnaB (R328A/R329A) produces almost entirely medium FRET states that are very dynamic, with a large number of transitions and relatively short dwell times indicative of severe destabilization of binding (Figure 2.12H &
Figure 2.13F). The R328A/R329A mutation exists around the ‘waist’ of the hexamer, where the N-terminal domain sits atop the C-terminal domain. In contrast to wild-type, very little high FRET signal from the R328A/R329A mutant on the 30/50 substrate is observed. These results indicate that R328 and R329 may be required to stabilize longer excluded strands (40 and 50(dT)) around the waist of the helicase and mediate interactions with other regions of the hexamer.
Altogether, these mutants alter the interaction between the excluded strand and the exterior of the helicase to varying degrees. The amount of destabilization or altered external DNA binding paths can depend on excluded strand length and provide information on the contacts all along the lateral length of the hexamer. As emphasized above, these results are similar to those obtained previously for SsoMCM on fork DNA substrates; where it was determined that disruption of the wrapping interaction caused greatly reduced unwinding
Figure 2-15: Summary of smFRET Wrapping Assays.
A homology model of E.coli DnaB is shown with each subunit of the hexamer colored separately. Mutated residues are colored in blue and labeled with summarizing descriptions of the effects of mutating each residue as determined by the results shown in Figure 2.14.