Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

Ibryashkina, Elena M.; Sasnauskas, Giedrius; Solonin, Alexander S.; Zakharova, Marina; Šikšnys, Virginijus

doi:10.1016/j.jmb.2009.01.048

Cited by 17 publications

(19 citation statements)

References 26 publications

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“…Cfr42I is a tetramer in solution, binds to two recognition sequences, and cleaves both sequences at once (21). Eco29kI, in contrast, purifies as a monomer in solution (371), but binds to its recognition sequence as a homodimer, and cleaves one recognition sequence at a time (372); Eco29kI also crystallizes with DNA as a homodimer (239); Cfr42I has not been crystallized.…”

Section: Introductionmentioning

confidence: 99%

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Type II restriction endonucleases—a historical perspective and more

2014

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This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.

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Section: Introductionmentioning

confidence: 99%

“…A small number of REases including Cfr42I (CCGC|GG), its isoschizomer Eco29kI (372), and Hpy188I (TCN|GA) (240), use a third class of catalytic site termed ‘GIY-YIG’ (26). The DNA co-crystal structures of Eco29KI and Hpy88I, both Type IIP homodimers, have been solved (239).…”

Section: Introductionmentioning

confidence: 99%

Type II restriction endonucleases—a historical perspective and more

2014

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“…Recently, more Type II restriction endonucleases, namely, KpnI [36], MnlI [37], Hpy99I [35], Eco31I [38], [39], HphI [40], SphI [41], PacI and others [42] are identified as containing this HNH motif through X-ray crystallography or sequence alignment/structural prediction. GIY-YIG endonucleases (including homing endonucleases I-TevI [43], [44], nucleotide excise repair enzyme UvrC and Type IIP REases Hpy188I [45] Eco29kI [46], [47] and Cfr42I [47], [48]) is proposed to adapt a similar catalytic mechanisms as HNH/His-Cys endonuclease except for the use of Tyr as the general base based on the structure of UvrC [49].…”

Section: Discussionmentioning

confidence: 99%

Cofactor Requirement of HpyAV Restriction Endonuclease

et al. 2010

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Background Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms.Principal FindingsWe have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity.Conclusions/SignificanceSome HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

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“…The mechanism must involve repositioning of a (presumably) single active site within the catalytic domain on substrate to perform two sequential nicking reactions, with the bottom (non-coding) strand nicked before the top (coding) strand (27,29). This mechanism is likely to be distinct from other enzymes that contain the GIY-YIG domain, including the restriction enzyme Cfr42I that functions as a tetramer (30), Eco29kI that functions as a dimer (31), or the UvrC proteins that nick only a single-strand adjacent to a damaged base (21). In an effort to gain insight into the mechanism by which GIY-YIG homing endonucleases introduce a DSB, we have been studying I-BmoI (Figure 1), (32).…”

Section: Introductionmentioning

confidence: 99%

A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI

Kleinstiver

Fernandes

Gloor

et al. 2010

Nucleic Acids Research

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Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG homing endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of ∼90 amino acids.

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Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

Cited by 17 publications

References 26 publications

Type II restriction endonucleases—a historical perspective and more

Type II restriction endonucleases—a historical perspective and more

Cofactor Requirement of HpyAV Restriction Endonuclease

A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI

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