Novel m4C modification in type I restriction-modification systems

Abstract

We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.

Structural basis underlying complex assembly and conformational transition of the type I R-M system

“…1A and 2A). This is in contrast to the previous understanding that the TRD domains (12,13) or the conserved IPLPPL motifs are the major interacting regions (14). The two helices of the M subunits adopt an antiparallel arrangement and form a four-helix bundle structure together with the coiled-coil region of the S subunit ( Fig.…”

“…One reason is that the characterization of type I systems has been relatively difficult because the position of DNA cutting is nonspecific. However, this situation has changed with the new singlemolecule real-time DNA sequencing technology, which can directly detect the DNA methylations (6,12). Another reason is the lack of high-resolution structural information about the complex assembly, intermolecular interaction, and the conformational transition between different states.…”

“…The S subunit determines the DNA recognition sequence, which consists of two independent DNA-binding domains (Target Recognition Domains, or TRDs) connected by two long alpha helices (Conserved Regions, or CRs). This results in a characteristic asymmetric, bipartite recognition sequence consisting of two half-sequences, each containing 2-5 base pairs separated by a gap with a fixed number of nonspecific DNA bases (6,12). For example, the EcoKI system recognizes the DNA sequence of 5′-AACNNNNNNGTGC-3′.…”

“…This feature is critical for the host cells to periodically change the REase specificity to overcome the threat of the viruses with modified target sequences, while changing the MTase specificity in exactly the same way to modify and protect the host DNA (6). It has been proposed that the M subunits bind mainly to the TRD domains (12,13) and the conserved "IPLPPL" regions connecting CRs and TRDs (14) in the S subunit to form the MTase complex. However, if this is the case, it is hard to understand why the type I R-M enzymes can still be properly assembled after exchanging the TRD domains among the different enzymes (6).…”

Vitamin B and Vitamin C Affect DNA Methylation and Amino Acid Metabolism in Mycobacterium bovis BCG

“…This suggests that m4C can promote gene transcription to some extent, but using mechanisms that are still unknown. The m4C modification was observed in type I R-M systems, suggesting that, in addition to m6A modifications, they can also use this modification for host protection (Morgan et al, 2016).…”