Alteration of Sequence Specificity of the Type II Restriction Endonuclease HincII through an Indirect Readout Mechanism

Abstract

High resolution x-ray crystal structures of protein-DNA complexes have revealed a rational basis for sequence specificity in the form of direct contacts between amino acid side chains and DNA bases in the major groove (1, 2). However, cases have been reported where specificity of a protein for a particular base pair (or base pairs) within its recognition sequence exists even when no direct contacts are seen between the protein and DNA (3-11). In these cases the specificity exhibited by the DNA-binding protein appears to be manifested by indirect means. This "indirect readout" of the DNA sequence may involve water-mediated contacts, contacts to the sugar-phosphate backbone, or the utilization of sequence-dependent DNA structure and/or deformability (3, 12).The crystal structure of the type II restriction endonuclease HincII reveals distortion from canonical B-form of its bound cognate DNA (13). HincII recognizes and cleaves the sequence GTYRAC, having degeneracy in its specificity for the center two nucleotides by allowing the presence of any pyrimidinepurine, i.e. TA, TG, CA, or CG. Sequence-specific contacts between the protein and DNA can explain this specificity. The contact to the center YR step (CG in the original crystal structure (13)) consists of a hydrogen bond from Asn-141 to the N7 of the purine base. Such a contact can occur when either G or A, but not C or T, occur in the fourth position of the recognition sequence. No contacts are made to the pyrimidine base. Typically, the high degree of specificity achieved by type II restriction endonucleases is due in part to the fact that contacts occur to both bases of each base pair. The question remains of whether or not one hydrogen bond to each purine of the center base pairs can explain the HincII specificity against sequences containing GTYYAC, GTRYAC, or GTRRAC.The crystal structure of wild type HincII bound to one of the three cognate DNA sequences, GTCGAC, shows that HincII distorts the bound DNA at three loci, two as a result of intercalation of the Gln-138 side chain into the DNA duplex just outside of its recognition sequence (Fig. 1A, light brown spacefilling atoms ϭ the recognition sequence, dark brown spacefilling atoms ϭ flanking DNA), and the third site at the center YR base pair (Fig. 1, A, red ϭ Y (C or T), and blue ϭ R (A or G), B, right). A hypothesis has been put forth that links the distortion of DNA at the center CG to indirect readout of this sequence by HincII. Furthermore, modeling of the DNA distortion revealed a connection between the Gln-138 intercalation and the center base pair distortion three base pairs away (13).The studies herein describe the characterization of HincII structure and function when glutamine 138 has been substituted with phenylalanine, Q138F. Preliminary velocity measurements showed that Q138F HincII favors TA over CG at the center YR step to a greater extent than wild type. Subsequent DNA binding assays and single turnover cleavage rate measurements revealed that the alteration in specificity toward cognate ...

DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target

“…Both of those enzymes are also homodimeric, Type IIP restriction endonucleases that recognize and cleave palindromic DNA target sites and also produced products with blunt ends. The more closely related of these two proteins, HincII, displays approximately a 3 Å backbone rmsd over 251 aligned residues, when using PDB ID 2gih (26) for comparison. Those two enzymes share 33 amino acids in common, corresponding to 14% sequence identity.…”

Early Interrogation and Recognition of DNA Sequence by Indirect Readout

“…Using this assay, the N141A HincII enzyme does not show any preference among the three HincII cognate sequences, with rate constants of 8.8±1.3×10 -5 s -1 (3.2±0.5×10 -1 hr -1 ), 9.2±3.9×10 -5 s -1 (3.3±1.4×10 -1 hr -1 ), and 11.0±4.1×10 -5 s -1 (4.0±0.2×10 -1 hr -1 ) for DNA containing the HincII sequences GTTAAC, GTCGAC, and the nonpalindromic GTTGAC/GTCAAC, respectively. N141A HincII DNA cleavage activity is approximately 1000 fold reduced relative to that of wild type HincII (Joshi et al, 2006). No cleavage was found in assays with the Noncognate GC DNA after 24 hours incubation (data not shown).…”

Probing the Run-On Oligomer of Activated SgrAI Bound to DNA

“…Second, in the absence of activation the cleavage of the second type (40-2B, Table 1) is much slower in one strand than the other (slower in the bottom strand, Table 1). This may be because on this strand the cleavage site is closer to the substitution in the sequence (the difference in the sequence compared to the primary site sequence, Fig 1), hence potentially resulting in greater structural disruption of the cleavage active site, a phenomenon we have seen before with a different restriction endonuclease [25,26]. It is interesting that the differences in the rates of cleavage of the two strands disappear with higher concentrations of activator DNA (Table 1).…”