Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA
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eLife assessment
This manuscript highlights single-stranded DNA exo- and endo-nuclease activities of ExoIII as a potential caveat and an underestimated source of decreased efficiency in its use in biosensor assays. The data present convincing evidence for the ssDNA nuclease activity of ExoIII and identifies residues that contribute to it. The findings are useful, but the study remains incomplete as the effect on biosensor assays was not established.
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Abstract
Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3′→5′ exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII ( Escherichia coli ) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labelled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5′-bond of phosphodiester from 3′ to 5′ end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3′-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.
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eLife assessment
This manuscript highlights single-stranded DNA exo- and endo-nuclease activities of ExoIII as a potential caveat and an underestimated source of decreased efficiency in its use in biosensor assays. The data present convincing evidence for the ssDNA nuclease activity of ExoIII and identifies residues that contribute to it. The findings are useful, but the study remains incomplete as the effect on biosensor assays was not established.
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors show compelling data indicating that ExoIII has significant ssDNA nuclease activity that is posited to interfere with biosensor assays. This does not come as a surprise as other published works have indeed shown the same, but in this work, the authors provide a deeper analysis of this underestimated activity.
Strengths:
The authors used a variety of assays to examine the ssDNA nuclease activity of ExoIII and its origin. Fluorescence-based assays and native gel electrophoresis, combined with MS analysis clearly indicate that both commercial and laboratory purified ExoIII contain ssDNA nuclease activity. Mutational analysis identifies the residues responsible for this activity. Of note is the observation in this submitted work that the sites of ssDNA and dsDNA …
Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors show compelling data indicating that ExoIII has significant ssDNA nuclease activity that is posited to interfere with biosensor assays. This does not come as a surprise as other published works have indeed shown the same, but in this work, the authors provide a deeper analysis of this underestimated activity.
Strengths:
The authors used a variety of assays to examine the ssDNA nuclease activity of ExoIII and its origin. Fluorescence-based assays and native gel electrophoresis, combined with MS analysis clearly indicate that both commercial and laboratory purified ExoIII contain ssDNA nuclease activity. Mutational analysis identifies the residues responsible for this activity. Of note is the observation in this submitted work that the sites of ssDNA and dsDNA exonuclease activity overlap, suggesting that it may be difficult to identify mutations that affect one activity but not the other. In this regard, it is of interest the observation by the authors that the ssDNA nuclease activity depends on the sequence composition of the ssDNA, and this may be used as a strategy to suppress this activity when necessary. For example, the authors point out that a 3′ A4-protruding ssDNA could be employed in ExoIII-based assays due to its resistance to digestion. However, this remains an interesting suggestion that the authors do not test, but that would have strengthened their conclusion.
Weaknesses:
The authors provide a wealth of experimental data showing that E. coli ExoIII has ssDNA nuclease activities, both exo- and endo-, however this work falls short in showing that indeed this activity practically interferes with ExoIII-driven biosensor assays, as suggested by the authors. Furthermore, it is not clear what new information is gained compared to the one already gathered in previously published works (e.g. references 20 and 21). Also, the authors show that ssDNA nuclease activity has sequence dependence, but in the context of the observation that this activity is driven by the same site as dsDNA Exo, how does this differ from similar sequence effects observed for the dsDNA Exo? (e.g. see Linxweiler, W. and Horz, W. (1982). Nucl. Acids Res. 10, 4845-4859).
Because of the claim that the underestimated ssDNA nuclease activity can interfere with commercially available assays, it would have been appropriate to test this. The authors only show that ssDNA activity can be identified in commercial ExoIII-based kits, but they do not assess how this affects the efficiency of a full reaction of the kit. This could have been achieved by exploiting the observed ssDNA sequence dependence of the nuclease activity. In this regard, the work cited in Ref. 20 showed that indeed ExoIII has ssDNA nuclease activity at concentrations as low as 50-fold less than what test in this work. Ref 20 also tested the effect of the ssDNA nuclease activity in Targeted Recycle Assays, rather than just testing for its presence in a kit.
Because of the implication that the presence of ssDNA exonuclease activity may have in reactions that are supposed to only use ExoIII dsDNA exonuclease, it is surprising that in this submitted work no direct comparison of these two activities is done. Please provide an experimental determination of how different the specific activities for ssDNA and dsDNA are.
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Reviewer #2 (Public Review):
Summary:
This paper describes some experiments addressing 3' exonuclease and 3' trimming activity of bacterial exonuclease III. The quantitative activity is in fact very low, despite claims to the contrary. The work is of low interest with regard to biology, but possibly of use for methods development. Thus the paper seems better suited to a methods forum.
Strengths:
Technical approaches.
Weaknesses:
The purity of the recombinant proteins is critical, but no information on that is provided. The minimum would be silver-stained SDS-PAGE gels, with some samples overloaded in order to detect contaminants.
Lines 74-76: What is the evidence that BER in E. coli generates multinucleotide repair patches in vivo? In principle, there is no need for the nick to be widened to a gap, as DNA Pol I acts efficiently from a …
Reviewer #2 (Public Review):
Summary:
This paper describes some experiments addressing 3' exonuclease and 3' trimming activity of bacterial exonuclease III. The quantitative activity is in fact very low, despite claims to the contrary. The work is of low interest with regard to biology, but possibly of use for methods development. Thus the paper seems better suited to a methods forum.
Strengths:
Technical approaches.
Weaknesses:
The purity of the recombinant proteins is critical, but no information on that is provided. The minimum would be silver-stained SDS-PAGE gels, with some samples overloaded in order to detect contaminants.
Lines 74-76: What is the evidence that BER in E. coli generates multinucleotide repair patches in vivo? In principle, there is no need for the nick to be widened to a gap, as DNA Pol I acts efficiently from a nick. And what would control the extent of the 3' excision?
Figure 1: The substrates all report only the first phosphodiester cleavage near the 3' end, which is quite a limitation. Do the reported values reflect only the single phosphodiester cleavage? Including the several other nucleotides likely inflates that activity value. And how much is a unit of activity in terms of actual protein concentration? Without that, it's hard to compare the observed activities to the many published studies. As best I know, Exo III was already known to remove a single-nucleotide 3'-overhang, albeit more slowly than the digestion of a duplex, but not zero! We need to be able to calculate an actual specific activity: pmol/min per µg of protein.
Figures 2 & 3: These address the possible issue of 1-nt excision noted above. However, the question of efficiency is still not addressed in the absence of a more quantitative approach, not just "units" from the supplier's label. Moreover, it is quite common that commercial enzyme preparations contain a lot of inactive material.
Figure 4D: This gets to the quantitative point. In this panel, we see that around 0.5 pmol/min of product is produced by 0.025 µmol = 25,000 pmol of the enzyme. That is certainly not very efficient, compared to the digestion of dsDNA or cleavage of an abasic site. It's hard to see that as significant.
Line 459 and elsewhere: as noted above, the activity is not "highly efficient". I would say that it is not efficient at all.
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Reviewer #3 (Public Review):
Overall:
ExoIII has been described and commercialized as a dsDNA-specific nuclease. Several lines of evidence, albeit incomplete, have indicated this may not be entirely true. Therefore, Wang et al comprehensively characterize the endonuclease and exonuclease enzymatic activities of ExoIII on ssDNA. A strength of the manuscript is the testing of popular kits that utilize ExoIII and coming up with and testing practical solutions (e.g. the addition of SSB proteins ExoIII variants such as K121A and varied assay conditions).
Comments:
(1) The footprint of ExoIII on DNA is expected to be quite a bit larger than 5-nt, see structure in manuscript reference #5. Therefore, the substrate design in Figure 1A seems inappropriate for studying the enzymatic activity and it seems likely that ExoIII would be interacting …
Reviewer #3 (Public Review):
Overall:
ExoIII has been described and commercialized as a dsDNA-specific nuclease. Several lines of evidence, albeit incomplete, have indicated this may not be entirely true. Therefore, Wang et al comprehensively characterize the endonuclease and exonuclease enzymatic activities of ExoIII on ssDNA. A strength of the manuscript is the testing of popular kits that utilize ExoIII and coming up with and testing practical solutions (e.g. the addition of SSB proteins ExoIII variants such as K121A and varied assay conditions).
Comments:
(1) The footprint of ExoIII on DNA is expected to be quite a bit larger than 5-nt, see structure in manuscript reference #5. Therefore, the substrate design in Figure 1A seems inappropriate for studying the enzymatic activity and it seems likely that ExoIII would be interacting with the FAM and/or BHQ1 ends as well as the DNA. Could this cause quenching? Would this represent real ssDNA activity? Is this figure/data necessary for the manuscript?
(2) Based on the descriptions in the text, it seems there is activity with some of the other nucleases in 1C, 1F, and 1I other than ExoIII and Cas12a. Can this be plotted on a scale that allows the reader to see them relative to one other?
(3) The sequence alignment in Figure 2N and the corresponding text indicates a region of ExoIII lacking in APE1 that may be responsible for their differences in substrate specificity in regards to ssDNA. Does the mutational analysis support this hypothesis?
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