A Unique m6A-Dependent Restriction Endonuclease from an Archaeal Virus

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.