Abstract
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. Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5′-C(mC)DG-3′ target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G). Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy. 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.
Highlights
The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modificationdependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts
In the present study we report the crystal structure of the N-terminal DNA binding domain of the LpnPI restriction endonuclease (LpnPI-N), which recognizes the modified cytosine in the sequence context 5 -C(mC)DG-3, and provide mutational/loop-swapping analysis that supports the structural model for the sequence recognition
The largest contact surface (∼750 A 2) is formed between the N- and C-termini of both A and B subunits that encircle the 160–170 -hairpins of the symmetry related A/B subunits (Supplementary Figure S3). This ‘pinching’ interaction, is not functionally important, since LpnPI-N is a monomer in solution (Supplementary Figure S4)
Summary
The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modificationdependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts. We report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5 -C(mC)DG-3 target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G). MspJI family enzymes recognize 5mC and 5hmC in various sequence contexts and cut both DNA strands 12/16 nt downstream of the modified cytosine They are arranged as the N-terminal SRA-like domain and the C-terminal PD-(D/E)XK nuclease domain fusions [11,12,13].
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