Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family

Abstract

Restriction endonucleases (REs) of the CCGG-family recognize a set of 4–8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5′-CCNGG flip the central N nucleotides and ‘compress’ the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5′-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not ‘compress’ the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.

Web 3DNA 2.0 for the analysis, visualization, and modeling of 3D nucleic acid structures

“…At the time of this writing, the server has been cited over 230 times (including 34 times in 2018), according to Google Scholar. Recent citations to w3DNA include: calculations of the DNA helical axis and twist angles (17), determination of the conformational parameters of DNA bound to PfoI and other CCGG-family restriction endonucleases (18), and generation of initial atomistic duplex and triplex models (19,20) for molecular dynamics simulations.…”

EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates

“…Type IIE EcoRII restriction endonuclease belongs to the CCGG-family of restriction enzymes that recognize target sites containing 5'-CCGG or 5'-CCNGG sequence and cut it before the outer C [18]. EcoRII, PspGI, Ecl18kI and PfoI that interact with 5'-CCNGG flip nucleotides at the center of pseudopalindromic target sequences [10,14,15,18,19]. Then EcoRII, PspGI and Ecl18kI compress DNA to make the 5 bp 5'-CCNGG sequence imitating a 4 bp 5'-CCGG sequence.…”

“…The CCGG-family of restriction endonucleases share a common mechanism of the CCGG tetranucleotide recognition [18]: the N-terminus of a conserved α-helix lays into DNA major groove and conserved amino acid residues R-(D/E)R form base specific contacts to carbonyl and amino groups, and to N7 atoms of CCGG bases. In the case of EcoRII conserved R-(D/E)R motif includes К 328 , D 329 and R 330 (KDR motif) [18]. We revealed the following contacts from crystal structure of EcoRII-C/ DNA complex (PDB file 3HQG [10]) using Chimera 1.14.…”