The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein

Abstract

Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.

Structure and operation of the DNA-translocating type I DNA restriction enzymes

“…SAXS and SANS data previously showed that the EcoR124I MTase collapsed from an extended structure to a more compact form in the presence of DNA (Taylor et al 1994(Taylor et al , 2010, as also shown by the almost complete protection from proteolysis when the MTase was bound to DNA (Webb et al 1995). The recent EM analysis of the EcoKI MTase also suggested an opening and closing cycle for DNA binding but of lesser magnitude than for EcoR124I (Kennaway et al 2009). …”

“…4E; Walkinshaw et al 2002). This orientation of Ocr in the EM map, coupled with the structural models of the MTase core of EcoKI (Kennaway et al 2009) and of EcoR124I (based on that of EcoKI as described in the Supplemental Material; Supplemental Fig. S4A), allows only one possible orientation of the MTase atomic models within the EM envelope (Fig.…”

“…The structures show EcoKI bound to a DNA duplex or to a DNA mimic anti-restriction protein, and EcoR124I in the absence and presence of a DNA duplex or an antirestriction protein. With the aid of numerous biochemical constraints, known crystallographic structures of type I RM subunits, the location of the HsdS and HsdM within the MTase at low resolution (Callow et al 2007;Taylor et al 2010), and our recent reconstruction of the core MTase structure (Kennaway et al 2009), we can construct a unique arrangement of the subunits within the EM structures, rationalize all previous knowledge about these complex machines, and reveal an evolutionary link between type I and type II RM systems.…”

“…In contrast to the situation with type II restriction enzymes, it is only very recently that partial atomic structures for type I RM subunits have emerged (Calisto et al 2005;Kim et al 2005;Obarska et al 2006;Obarska-Kosinska et al 2008;Kennaway et al 2009;Lapkouski et al 2009;Uyen et al 2009;Taylor et al 2010;Gao et al 2011), and their mechanisms, which most dramatically involve the translocation of thousands of base pairs of DNA with the formation of supercoiled loops (Yuan et al1980;Endlich and Linn 1985;Studier and Bandyopadhyay 1988;García and Molineux 1999), still present many questions. The type I RM enzymes are complex structures with two HsdR restriction (R) subunits, two HsdM modification (M) subunits, and one HsdS sequence specificity (S) subunit (Murray 2000;Loenen 2003), each with a number of domains (Supplemental Fig.…”

“…The HsdS structure displays pseudo twofold symmetry (Calisto et al 2005;Kennaway et al 2009;Kim et al 2005;Gao et al 2011), and this symmetry is imposed on the whole structure of these enzymes (Kneale 1994;Dryden et al 1995;Davies et al 1999;Kennaway et al 2009). The two HsdM lie on either side of the HsdS to form the M 2 S 1 methyltransferase (MTase) core.…”