Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI

Roey, P. Van; Meehan, Lisa; Kowalski, Joseph C.; Belfort, Marlene; Derbyshire, Victoria

doi:10.1038/nsb853

Cited by 68 publications

(128 citation statements)

References 30 publications

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“…However, alternative schemes for making double-strand breaks in DNA have yet to be eliminated for I-TevI (21). The alternatives could perhaps be tested by examining I-TevI reactions on duplexes attached to magnetic beads, on transferring the beads from nonpermissive to permissive conditions (namely Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The intron-encoded endonuclease I-TevI is a monomer with one active site and it has been suggested that, after it has cleaved the bottom strand, it distorts the DNA to access the top strand (20). However, this scheme has yet to be confirmed: given the crystal structure of the catalytic domain of I-TevI, both transient dimerization and hairpin schemes are also possible (21).…”

mentioning

confidence: 99%

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How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Sasnauskas

Halford

Šikšnys

2003

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

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Unlike other restriction enzymes, BfiI functions without metal ions. It recognizes an asymmetric DNA sequence, 5 -ACTGGG-3 , and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like FokI, dimerize transiently during catalysis. BfiI is also a dimer but it has only one active site, at the dimer interface. We show here that BfiI remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and͞or the DNA in the BfiI-DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting BfiI into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5 phosphate in the bottom strand. BfiI thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.S equence-specific cleavage of double-stranded DNA underpins many genetic events, including recombination, transposition, viral DNA integration, and the restriction of DNA (1-4). The enzymes that make double-strand breaks use many different strategies to achieve this end. The simplest may be the orthodox type II restriction enzymes such as EcoRI or EcoRV, dimers of identical subunits that recognize palindromic DNA sequences. In the presence of Mg 2ϩ , they cleave both strands at fixed positions in their recognition sites (3). They bind their target sites symmetrically, so that one active site from the dimer is positioned against one DNA strand and likewise the second active site on the other strand. Independent reactions in each active site then generate the double-strand break. Some homing endonucleases, such as I-CreI, also act in this way (4), as do the enzymes that resolve Holliday junctions (2).This strategy cannot apply to enzymes that recognize nonpalindromic sites and cut both strands away from the recognition site (3). For example, FokI, a type IIS restriction enzyme, recognizes the sequence 5Ј-GGATG-3Ј and cuts top and bottom strands 9 and 13 nt downstream of this site, respectively (5). FokI is a monomer with separate domains for DNA recognition and catalysis. The catalytic domain has a single active site, which is like that in each subunit of an orthodox enzyme, so the FokI monomer cannot cleave both DNA strands (6). To make doublestrand breaks, the monomer of FokI bound to its recognition site associates transiently with a second monomer to form a dimer with two active sites (7-9). Many type IIS enzymes operate in this way (10,11).An alternative to using a homodimeric enzyme to make double-strand breaks is an enzyme with different subunits. For example, the transposition of Tn7 requires two proteins, TnsA and TnsB, that each cleave one strand of the DNA (12). Interestingly, the structure of TnsA is similar...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Sasnauskas

Halford

Šikšnys

2003

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

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“…However, several active GIY-YIG ENs (i.e., all Seg proteins) do not contain either motif B with the invariant Arg or any other positively charged amino acids in this region, which argues against the indispensable role of this Arg (16,33). The neighboring highly conserved H761 may be involved in catalysis instead, as suggested for I-TevI (17).…”

Section: Discussionmentioning

confidence: 90%

“…Notably, helix 1 exhibits a strong positive charge in both molecules, with side chains of five lysine residues exposed on the surface of the Penelope EN. This feature is thought to be responsible for interaction with DNA through phosphate backbone contacts (17). The conserved N816 at the C terminus is able to form hydrogen bonds with I747 in the backbone of ␤-strand 2, thus, possibly keeping helix 3 in place.…”

Section: Sequence Conservation Of Rt and En Domainsmentioning

confidence: 99%

“…5 Lower) shows that it shares with other members of the GIY-YIG family the most conserved residues thought to constitute the active site (5,16,17). In particular, the Penelope EN contains residues (Y730, Y746, G748, R757, E800, and N816) presumably corresponding to invariant or highly conserved residues required for catalytic activity of the prototype GIY-YIG EN I-TevI from bacteriophage T4 (Y6, Y17, G19, R27, E75, and N90).…”

Section: Sequence Conservation Of Rt and En Domainsmentioning

confidence: 99%

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Reverse transcriptase and endonuclease activities encoded by Penelope -like retroelements

Pyatkov

Arkhipova

Malkova

et al. 2004

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

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Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn 2؉ and Mg 2؉ ). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon.GIY-YIG endonuclease ͉ retrotransposons ͉ Drosophila virilis ͉ Uri domain

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Homing Endonucleases: From Genetic Anomalies to Programmable Genomic Clippers

Belfort

Bonocora

2014

Methods in Molecular Biology

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Homing endonucleases are strong drivers of genetic exchange and horizontal transfer of both their own genes and their local genetic environment. The mechanisms that govern the function and evolution of these genetic oddities have been well documented over the past few decades at the genetic, biochemical, and structural levels. This wealth of information has led to the manipulation and reprogramming of the endonucleases and to their exploitation in genome editing for use as therapeutic agents, for insect vector control and in agriculture. In this chapter we summarize the molecular properties of homing endonucleases and discuss their strengths and weaknesses in genome editing as compared to other site-specific nucleases such as zinc finger endonucleases, TALEN, and CRISPR-derived endonucleases.

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Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI

Cited by 68 publications

References 30 publications

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

Reverse transcriptase and endonuclease activities encoded by Penelope -like retroelements

Homing Endonucleases: From Genetic Anomalies to Programmable Genomic Clippers

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