Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities

Abstract

To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2′-O-methyl nucleotide (2′-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2′-O-methyl nucleotide has a C3′-endo sugar pucker conformation different from the C2′-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2′-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2′-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2′-fluoro nucleic acid (2′-FNA) and locked nucleic acid (LNA) having similar C3′-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2′-endo to C3′-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2′-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

“…S9, E to G). These kinetic properties are similar to those of other PD-D/E(X)K nucleases, such as Eco RI (27, 33). MIF also cleaved dsPS 30 (fig.…”

MIF is a 3’ flap nuclease that facilitates DNA replication and promotes tumor growth

“…Moreover, the unique nuclease selectivity of purified MIF protein on various DNA substrates with different structure or length of nucleotides at the 3' end reinforces the selectivity and potency of MIF's 3' nuclease activity. Although the kinetic analysis of MIF activity revealed a relatively slow reaction with t 1/2 at 7.7-10.8 min, K M at 0.7-0.9 μM and k cat at 0.02/min, it is comparable to some well-recognized nucleases including MutsS2, exoribonuclease (ExoN), ribozyme, and EcoRI [43][44][45][46][47] . For example, the nuclease activity of Thermus thermophilus MutS2 (ttMutS2), which is known to play an important role in MMR, has been reported to have a K M at 290 nM and k cat at 0.041/ min 44 , which are comparable to those of MIF.…”

“…For example, the nuclease activity of Thermus thermophilus MutS2 (ttMutS2), which is known to play an important role in MMR, has been reported to have a K M at 290 nM and k cat at 0.041/min 44 , which are comparable to those of MIF. The restriction nuclease EcoRI’s t 1/2 is 9.7 min calculated by UV linear dichroism 46 and about 8 min monitored by fluorescence changes 47 . As with many nucleases, it is possible that the additional enhancer may help MIF bind and cleave DNA efficiently in vivo, which requires further investigation.…”

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases

“…2′-F RNA and LNA locks sugar ring in C3′ endopucker conformation and has many advantages such as increasing affinity to RNA target 5,37) and protecting oligonucleotide against nuclease digestion 38) . In our previous study, we also found their applications in regulating endonuclease cleavage efficiency 39,40) . Though 2′-OMeRNA has similar characteristics of the C3′ endopucker conformation, it should be avoided in RCA application since it strongly suppressed RCA efficiency.…”