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Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.

ABSTRACT: The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.

SUBMITTER: Lambert AR

PROVIDER: S-EPMC2390919 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.

Lambert Abigail R AR Sussman Django D Shen Betty B Maunus Robert R Nix Jay J Samuelson James J Xu Shuang-Yong SY Stoddard Barry L BL

Structure (London, England : 1993) 20080401 4

The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions n ...[more]

PMID: 18400177

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Project description:Prokaryotes possess numerous diverse defense systems to resist viral infections, while some viruses have also evolved antiviral defense systems to exclude other viruses in cases of multiple infections. Here, we report the first virus-derived modification-dependent restriction endonuclease (HHPV4I) from the archaeal virus HHPV4 (Haloarcula hispanica pleomorphic virus 4). HHPV4I contains an SRA domain, a winged helix (wH) domain, and an HNH domain; recognizes the Gm6ATC site; and specifically binds to Gm6ATC site-containing DNA. Both the wH domain and the HNH domain are responsible for DNA binding. Unlike the well-known m6A-specific restriction enzyme DpnI, HHPV4I only efficiently cleaves DNA with a fully methylated Gm6ATC site and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. Furthermore, HHPV4I preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC site. Thus, the cleavage pattern of HHPV4I is distinct from those of all of the presently characterized restriction endonucleases. Mutations in the wH domain of HHPV4I do not alter m6A-dependent endonuclease activity, but they decrease recognition sequence specificity, thus expanding the cleaving capacity to more m6A-containing DNA sequences. The wH domain provides a target for searching, developing, and engineering novel m6A-dependent endonucleases. IMPORTANCE Many modification-dependent restriction endonucleases (MDREs) were identified in prokaryotes and recognized modified cytosine bases, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and glucosyl-5-hydroxymethylcytosine (g5hmC). The first virus-derived MDRE (HHPV4I) from the archaeal virus HHPV4 was identified in this study. The viral MDRE suggested a new strategy employed by the virus to exclude other viruses in the case of multiple replications. HHPV4I is a novel N6-methyladenine (m6A)-dependent restriction endonuclease, while the cleavage pattern of HHPV4I is distinct from the well-known m6A-dependent restriction endonuclease DpnI. HHPV4I recognizes Gm6ATC sites and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. It preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC sites. Furthermore, mutations in the HHPV4I wH domain can alter the sequence specificity without impeding the m6A-dependent DNA cleavage activity, providing a target for engineering more m6A-dependent endonucleases with different sequence specificities.

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Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.

Publications

Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.

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