To rule out the possibility that the appearance of two unfolding transitions of MszExo I was caused by increased ionic strength of buffers with 5 mM MgCl 2 or 20 mM MgCl 2 , DSC measurements were performed as a function of salt concentration Fig.
As the figure shows, MszExo I always exhibited single peak during thermal unfolding in buffers with different concentration of salts, though higher T m value with increased salt concentration was also observed Fig.
However, in the absence of sodium chloride, the unfolding transition became less cooperative Fig. Taken together, the role of magnesium ions in thermal unfolding of MszExo I is unique and could not be replaced by salts. Exo I was first characterized in E. It is conserved in bacteria, but not in archaea and eukarya. Recently, a hyperthermophilic exonuclease I, PfuExo I, was identified, though with limited sequence similarity to E.
It digests ssDNA at every two nucleotides, different from the catalytic mechanism of E. Considering this fact, moderately thermophilic bacteria seemed a productive source for finding robust alternatives to E. In bacteria, the nucleic acids metabolic pathways of E.
However, few reports on the DNA metabolic pathways of moderately thermophilic bacteria have been published, probably due to the small population of moderately thermophilic bacteria being found. However, more and more moderately thermophilic bacteria have been described recently, in many cases as potential sources of useful enzymatic activities [ 24 — 28 ]. For example, the keratinolytic protease from Meiothermus ruber H shows extraordinary tolerance to denaturants [ 28 ].
Several genomes of moderately thermophilic bacteria have been completely sequenced [ 29 , 30 ]. Therefore, more studies of moderately thermophilic bacteria are expected. Our report is the first characterizing an enzyme involved in a DNA repair system from a moderately thermophilic bacteria. At higher temperatures the amount of DNA damage will increase, hence repair systems are required to be more efficient to ensure the faithful transmission of genomic DNA to the next generation.
More research is needed to elucidate the differences in DNA repair systems from moderately thermophilic bacteria and mesophilic ones, as well as the similarities. In our study, we found that MszExo I exhibits similar metal ion binding affinity and specific activity as E. However, during thermal denaturation different unfolding pathways were observed even though their crystal structures are highly conserved.
This may be related to the high performance of MszExo I at moderately thermophilic temperatures. Residues at the active site are highlighted in yellow, and those at the anchor site are highlighted in red. The yield of MszExo I was around 1—2 mg per liter culture. Marker, color protein standard broad range New England Biolabs. We would like to thank Professor Victor L. Davidson from University of Central Florda, Dr. Analyzed the data: LF RM. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense , a moderately thermophilic methanotroph. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: MszExo I gene sequence is available from the genebank database accession number: KM Protein expression and purification The E.
Crystallization and structure determination For crystallization studies the MszExo I gene was synthesized with an N-terminal cleavable strep tag. Download: PPT. Determination of metal ion binding affinity K obs,Mg of E. Effect of temperature on exonuclease activity Activities of E. Thermal inactivation assay Enzymes were diluted in exonuclease reaction buffer to a final concentration of 15 nM. Fig 3. Structural alignment between E. Similar metal ion binding affinity is observed in MszExo I and E.
Fig 4. Determination of the metal ion binding affinity K obs,Mg of E. Broader working temperature and higher thermal stability of MszExo I MszExo I is predicted to have better thermal stability than its E.
Fig 5. Thermal profiling of E. Fig 6. Thermal stability of E. Magnesium ion modulates thermal unfolding curve of MszExo I To further investigate the unfolding transitions of MszExo I, DSC measurements were performed as a function of concentration of magnesium ions. Fig 7. Discussion Exo I was first characterized in E. Supporting Information. S1 File. Preparation of MszExo I protein for crystallization.
S1 Fig. Sequence alignment of E. S2 Fig. Pufication of MszExo I. Acknowledgments We would like to thank Professor Victor L. References 1. On the Specificity of Exonuclease I Phosphodiesterase. J Biol Chem — Nat Struct Biol 7: — Nucleic Acids Res — Busam RD Structure of Escherichia coli exonuclease I in complex with thymidine 5'-monophosphate.
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Explore Support Center Medical Affairs We provide medical information and facilitate research collaborations. Connect With Us. Local Sales Support Get in touch with a nearby distributor or sales representative. Find Sales Contact. Contact Us Customer Support. Add multiple products. Please Enquire This product is discontinued. Add to Helix This product is available through the Promega Helix onsite stocking program. Exonuclease III. References Weiss, B. Rogers, S. T5 Exonuclease also has ssDNA endonuclease activity.
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