Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI.
Bottom Line: The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modification-dependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts.Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI.Taken together, our results pave the way for specificity engineering of the cytosine modification-dependent restriction enzymes.
Affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania email@example.com.Show MeSH
Mentions: The flipped-out cytosine binding pockets are similar in all SRA domains (Figure 2). The cytosine 5-methyl group in MspJI pocket is in van der Waals distance from D117, Y114 and W101 residues, and apparently makes a weak C–H…O hydrogen bond to the carbonyl oxygen of G116. These interactions may serve to distinguish modified cytosine from an unmodified base (18). Equivalent positions in LpnPI and AspBHI are occupied by D85, Y82, W69, and G84 residues. The side walls of the MspJI pocket are formed by the residues W101, Y114 and D117, while D103, S90 and F115 make hydrogen bonds to the Watson-Crick edge of the modified cytosine. Equivalent residues in LpnPI are W69, Y82, D85, K87, D71, N63 and Y83 (W69, Y82, D85, R87, D71, N63, Y83 in AspBHI). Mutation of the AspBHI pocket residues D71, Y82 and D85 to alanine abolished the DNA cleavage activity (13).
Affiliation: Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania firstname.lastname@example.org.