NTP hydrolysis is the common mechanism all helicases use to generate energy
to translocate along single strand nucleic acids and catalyse strand separation. The hydrolytic reaction can be stimulated by the presence of nucleic acids, single or double stranded. Binding of the appropriate nucleic acid induces conformational changes that enable efficient NTP binding and hydrolysis. SsoCas3’ contains all the characteristic helicase motifs (I, II, VI) to catalyse NTP hydrolysis, so the ATPase activity of the protein was investigated by the malachite green colorimetric phosphate assay, as described in chapter 2. Wild-type and SsoCas3’ K46A (Walker A mutant)
protein (2 μΜ) was incubated in the presence or absence of ss- or ds DNA, RNA or
DNA/RNA heteroduplexes at temperatures ranging from 37oC - 65oC, in the presence
of 1 mM MgCl2. All nucleic acid substrates used were annealed or single-strand
oligonucleotides 25-40 nt in length (CRISPR-related substrates, tables 5.1, 5.2). Samples were taken across a 20 min time course and free phosphate levels were visualised with malachite green, a reagent producing a colour change upon interaction with free phosphate that can be monitored by absorbance at 650 nm. The intensity of absorbance is analogous to the levels of free phosphate in the sample, providing a qualitative assay to measure the rate of ATP hydrolysis. Results are summarised in figure 5.10.
Levels of ATP hydrolysis were significantly higher in the presence of ssDNA,
compared to ssRNA, dsDNA or dsRNA. Minimal levels of free phosphate were detected in the absence of nucleic acid, due to the fact that enzyme conformational flexibility may lead to a basal level of ATP hydrolysis (Soultanas & Wigley 2000). The mutation of the conserved lysine to alanine in the Walker A motif of SsoCas3’ would
render it incapable of hydrolysing ATP as this lysine interacts with the β-phosphate
and acts to stabilise the transition state of the hydrolytic reaction (Tuteja and Tuteja, 2004). Indeed, the SsoCas3’ K46A mutant exhibited basal background levels of ATP hydrolysis. This control also confirms that the ATPase activity observed is attributed to SsoCas3. Reaction rates, although extremely low compared to other helicases, were almost 5-fold higher when ssDNA was present, indicating an ssDNA-stimulated ATPase activity for SsoCas3’.
The effect of temperature on the ATPase reaction was also investigated, in
order to determine the optimum temperature range for protein function. Reactions
were carried out at 37oC, 45oC, 55oC and 65oC under identical conditions for 30 min
with 1 μΜ protein and in the presence of ssDNA. Control reactions with only ssDNA
were run in parallel to obtain the background levels of spontaneous ATP hydrolysis at
different temperatures, which revealed a basal level of 6.24 pmoles phosphate.min-1.
The levels of hydrolysed ATP increased with the temperature rise, with the optimum
temperature optimum is expected from an enzyme by a thermophilic organism such as S. solfataricus. Optimum growth for this archaeon is observed at 80oC, therefore it
would be expected that the enzyme would be more active approaching this
temperature. The fact that SsoCas3’ exhibits highest activity at 55oC could be related
to the fact that the context of Cas3’ function in vivo differs greatly from the minimum experimental set up presented here, as it is predicted to interact tightly with an HD- domain nuclease and potentially a CASCADE-like complex.
Considering that the natural Cas3’ substrates would most likely include an R-
loop, we also monitored the ATPase activity in the presence of a 25-base pair ds RNA- DNA heteroduplex. Reaction rates were comparable to the rates obtained in the presence of dsDNA and dsRNA, indicating that this type of substrate does not stimulate ATP hydrolysis. We can therefore infer that SsoCas3’ exhibits an ssDNA- dependent ATPase activity, in agreement with the results reported for the Streptococcus thermophilus Cas3 (Sinkunas et al. 2011). However, the reaction rates for SthCas3 were reasonably higher than SsoCas3, reflecting the processive helicase activity for this DExH-box protein. One explanation could be suggested considering the differences in ATP hydrolysis modes between DExH-box and DEAD-box proteins as outlined in section 5.1.2. Even though SsoCas3’ is a DExD-box family protein, it lacks the additional domains of SthCas3 that potentially needed to maintain a high rate of ATP hydrolysis, therefore mechanistically resembling DEAD-box proteins’ mode of action.
Figure 5.10: ATPase activity of WT and mutant SsoCas3’
(A) Course of ATP hydrolysis by SsoCas3’ in the presence of ssDNA, ssRNA and RNA-DNA hybrids at 55oC. A linear curve fit was applied to the data points. ATP hydrolysis in the presence of dsDNA and dsRNA was at the same levels as for dsRNA-DNA (not shown). ATPase activity although very low is clearly stimulated in the presence of ssDNA. Reaction rates in table (B) in pmoles phosphate. pmoles SsoCas3’ -1.min-1. (C) ATPase activity of
SsoCas3’ in the presence of ssDNA at different temperatures illustrated by pink blocks. Background levels of ATP hydrolysis in the presence solely of ssDNA are presented in blue blocks.
C. Temperature dependence on ATPase activity of Cas3’
Temp oC
rate of ATP hydrolysis +ssDNA 37oC 0.37 ± 0.08 45oC 0.59 ± 0.02 55oC 0.65 ± 0.01 65oC 0.33 ± 0.07 A. ATP hydrolysis at 55oC
Substrate rate of ATP hydrolysis WT
rate of ATP hydrolysis Walker A mutant - 0.06 ssDNA 0.34 ± 0.022 0.08±0.03 ssRNA 0.08 ± 0.019 dsRNA-DNA 0.08 ± 0.037 B