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Mutant Thermotoga maritima endonuclease V (Tma endoV M2-6)


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Background

Tma endoV M2-6 is an endonuclease having modified substrate specificity created by amino acid substitutions to Tma endoV (endonuclease V from thermophilic bacterium Thermotoga maritima). Tma endoV has a deoxyinosine 3'-endonuclease activity, i.e. it recognizes a hypoxanthine (the deoxyinosine base) and exhibits a nicking activity to hydrolyze the second phosphodiester bond to 3' the lesion base [1-4]. The wild-type endoV also shows nonspecific DNA cleaving activities, independent of the specific recognition of the lesion structure. Tma endoV M2-6 exhibits a significantly reduced nonspecific activity toward an intact DNA and retains the specific endonuclease activity against a deoxyinosine-containing DNA.

Optimum Temperature: 65°C

 CSR_EMV001_1  CSR_EMV001_2
Figure 1. Degradation of double-stranded DNA with Tma endoV M2-6
Upper panel : 12 Units of Tma endoV M2-6 was incubated with 217 ng of double-stranded DNA containing various contents of dI in 1 x Reaction Buffer (total volume 50 μl) at 65 Ž for indicated time. Lower panel : For comparison, the same amount of wild-type Tma endoV was incubated in the same conditions. Tma endoV M2-6 degraded only dI-containing DNA specifically, and exhibited no significant degradation of the normal DNA (not containing dI).
Figure 2. Cleavage time course of dI-containing double-stranded DNA.
1 Unit of Tma endoV M2-6 and 217 ng of dI-containing double-stranded DNA (dI-malB2) were incubated in 1 x Reaction Buffer (total volume 50 μl) at 65Ž. Time courses of DNA cleavage (%) by the fresh enzyme (closed circles) and by the preheated enzyme at 65 Ž for 1 hour (open boxes) are shown. Tma endoV M2-6 did not show significant loss of activity level even after the heat treatment at 65 Ž for 1 hour.
CSR_EMV001_3 CSR_EMV001_4
Figure 3. Degradation of single-stranded DNA with Tma endoV M2-6
Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. The carboxyfluorescein(FAM)-labeled DNA fragments were excited at 473 nm.
Figure 4. Relative preference of 5’ or 3’ neighboring base in dI recognition and cleavage by Tma endoV
*I-Vol ave. is the average signal intensity of each neighboring base to dI site for cleaved fragments from various sequence context.
References

1) Huang J, et al., Biochemistry, 40(30), 8738-8748 (2001).
2) Huang J, et al., Biochemistry, 41(26), 8342-8350 (2002).
3) Hitchcock TM, et al., Nucleic Acids Res, 32(13), 4071-4080 (2004).
4) Feng H, et al., Biochemistry, 44(34):11486-11495 (2005).
5) Nakashima K, Takano F, Matsuo K, Ohiso I, Highly specific recognition and
cleavage of deoxyinosine-containing DNA by mutant Thermotoga maritima
endonuclease V. The 32nd Annual Meeting of the Molecular Biology of Japan
(MBSJ2009).

Product List

Note : Product "CSR-EMV001" cannot be sold to USA because of the patent issue.


Product Name Cat# Quantity Price

Tma endoV M2-6 (mutant Thermotoga maritima endonuclease V) DataSheet ,

CSR-EMV001

100 UNIT N/A

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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