Mechanistic Insights from the Structures of HincII Bound to Cognate DNA Cleaved from Addition of Mg2+ and Mn2+

Etzkorn, C.; Horton, Nancy C.

doi:10.1016/j.jmb.2004.08.082

Cited by 19 publications

(26 citation statements)

References 60 publications

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“…4 and 9, C-D). Although two divalent cations are expected to bind in the case of Mg 2ϩ and as observed with Mn 2ϩ (17), Ca 2ϩ binds in only one of the two positions seen to be occupied by Mn 2ϩ in both wild type and Q138F HincII.…”

Section: Resultsmentioning

confidence: 84%

“…The divalent cation Ca 2ϩ inhibits DNA cleavage while increasing the affinity and specificity of type II restriction endonucleases for their cognate DNA (14 -16) and is found (pink ball, Fig. 1A) to bind very close to the identified Mg 2ϩ binding sites in the active sites of enzymes studied (17). Thus, Ca 2ϩ serves as a mimic of the in vivo cofactor Mg 2ϩ , allowing the observation of a pretransition state structure.…”

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confidence: 92%

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

Joshi

Etzkorn

Chatwell

et al. 2006

Journal of Biological Chemistry

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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 ...

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Section: Resultsmentioning

confidence: 84%

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confidence: 92%

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

Joshi

Etzkorn

Chatwell

et al. 2006

Journal of Biological Chemistry

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“…This mechanism is supported by various co-crystal structures of pre-reactive and post-reactive complexes (BamHI [154], BglI [155], NgoMIV [48] and PvuII [156]). HincII is a problematic case; in the presence of Ca 2+ only one metal ion is seen in the pre-reactive complex [157], whereas in the post-reactive complex two Mg 2+ or Mn 2+ ions are present [158]. The authors favour a two-metal ion mechanism.…”

Section: Two-metal Ion Mechanismmentioning

confidence: 99%

Type II restriction endonucleases: structure and mechanism

Pingoud

Fuxreiter

Pingoud

et al. 2005

CMLS, Cell. Mol. Life Sci.

441

494

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Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few restriction endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II restriction endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.

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“…This suggests that a sequential cleavage mechanism is a possibility to consider for some of the monomeric members of the LAGLI-DADG family. Other catalytic mechanisms based on 2 metal ions have been proposed for restriction enzymes such as HincII (27), whose substrate-and product-bound structures resemble those of I-DmoI active site, containing 1 Ca 2ϩ and 4 Mn 2ϩ , respectively. Two of these Mn 2ϩ are arranged in a similar disposition as in I-DmoI, including a site between them occupied by a water molecule.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Homing endonucleases, also known as meganucleases, are sequencespecific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.gene targeting ͉ genetics ͉ protein-DNA interactions ͉ X-ray crystallography M eganucleases are sequence-specific enzymes that recognize large (12-45 bp) DNA target sites. These enzymes are often encoded by introns or inteins behaving as mobile genetic elements. They produce double strand breaks (DSB) that get repaired by homologous recombination with an intron-or intein-containing gene, resulting in the insertion of the intron or intein in that particular site (1). Meganucleases are being used to stimulate homologous gene targeting in the vicinity of their target sequences with the aim of improving current genome engineering approaches, alleviating the risks due to the randomly inserted transgenes (2, 3).The use of meganuclease-induced recombination has long been limited by the repertoire of natural meganucleases. In nature, meganucleases are essentially represented by homing endonucleases, a family of enzymes encoded by mobile genetic elements whose function is to initiate DSB induced recombination events in a process referred to as homing (4). The probability of finding a homing endonuclease cleavage site in a chosen gene is extremely low. Thus, making artificial meganucleases with custom-made substrate specificity is an intense area of research.Sequence homology has been used to classify homing endonucleases into 5 families, the largest one having the conserved LAGLI-DADG sequence motif. Homing endonucleases containing one such motif function as homodimers. In contrast, homing endonucleases containing 2 motifs are single chain proteins (5, 6). Structural information for several members of the LAGLIDADG endonuclease family indicate that these proteins adopt a similar active conformation as homodimers or as monomers with 2 separate domains (7-9). The LAGLIDADG motifs form structurally conserved ␣-helices tightly packed at the center of the interdomain or intermonomer interface. The last acidic residue of this LAGLI-DADG motif part...

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Mechanistic Insights from the Structures of HincII Bound to Cognate DNA Cleaved from Addition of Mg2+ and Mn2+

Cited by 19 publications

References 60 publications

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

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

Type II restriction endonucleases: structure and mechanism

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

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