Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Abstract

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1).

Influence of metal ions (Zn2+, Cu2+, Ca2+, Mg2+and Na+) on the water coordinated neutral and zwitterionicl-histidine dimer

“…For instance, in Cys 2 His 2 , zif268 and TATA binding protein, the Zn 2+ ion makes four coordinations, two each with cysteine and histidine, respectively. 7,27 Furthermore, in order to bind DNA, a water molecule is coordinated with zinc. 28 From the abovementioned paragraphs, we see that zinc coordination is common with histidine in zinc nger protein.…”

The metal dependence of single-metal mediated phosphodiester bond cleavage: a QM/MM study of a multifaceted human enzyme

“…[38][39][40] The preference for Mg 2+ and retention of activity in the presence of other metals correlates with the behaviour reported for other proposed singlemetal dependent nucleases (Moraxella nonliquefaciens restriction endonuclease II (MnII), group I intron-encoded endonuclease from Physarum polycephalum (I-Ppol) and HNH homing endonuclease (I-Hmul)), [41][42][43] while other potential enzymes in this class exhibit enhanced activity with transition metals (Klebsiella pneumoniae restriction endonuclease (Kpn1), Enterobacter hormaeche restriction endonuclease (Ehol), and Helicobacter pylori restriction endonuclease (HpyAV)). [44][45][46] In contrast to transition metals, APE1 activity is completely abolished in the presence of Ca 2+ . [38][39][40] The replacement of Mg 2+ by Ca 2+ has been shown to inhibit catalysis and permit crystallization of many two-metal dependent nucleases, [47][48][49] a concept supported by computational work.…”

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

“…These enzymes are not only important to protect bacterial cells from invasion by viral DNA, [ 1–3 ] but also widely used as genetic tools. [ 4,5 ] The REs require metal ions as cofactor for their activities, Mg 2+ being the natural cofactors for majority of enzymes, like EcoAI, [ 6 ] EcoRI, [ 7 ] EcoRV, [ 7–10 ] SepMI, [ 10,11 ] EhoI [ 10,11 ] to name only a few. Although REs can bind to other divalent metal ions, the protein has specific binding [ 11 ] to DNA in presence of both Ca 2+ and Mg 2+ .…”

“…[ 12,13 ] Numerous studies have been performed to understand role of Mg 2+ as natural cofactor. [ 11,14–17 ] But the microscopic origin of the specificity of metal ion cofactor Mg 2+ vis‐à‐vis Ca 2+ in DNA cleavage is unknown. [ 18–20 ]…”

“…performed to understand role of Mg 2+ as natural cofactor. [11,[14][15][16][17] But the microscopic origin of the specificity of metal ion cofactor Mg 2 + vis-à-vis Ca 2+ in DNA cleavage is unknown. [18][19][20] Naturally occurring restriction endonucleases are classified as four types such as type I, II, III and IV, depending on their structure, recognition site, cleavage site etc.…”