Structure of the motor subunit of type I restriction-modification complex EcoR124I

Abstract

Type I restriction-modification enzymes act as conventional adenine methylases on hemimethylated DNAs, but unmethylated recognition targets induce them to translocate thousands of base pairs before cleaving distant sites nonspecifically. The first crystal structure of a type I motor subunit responsible for translocation and cleavage suggests how the pentameric translocating complex is assembled and provides a structural framework for translocation of duplex DNA by RecA-like ATPase motors.

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

“…This model would suggest that the partially assembled R 1 M 2 S 1 form of EcoR124I would be able to cleave DNA. However, no cleavage was observed for this partially assembled enzyme , and thus the Lapkouski et al (2009) model cannot be entirely correct. The current model suggests that the endonuclease domain of one HsdR is in proximity to DNA translocated by the other HsdR (Fig.…”

“…Although the current models and their model share much in common, there are two main differences; namely, the orientation of the HsdR with respect to the MTase core, and the path taken by the DNA. Previously (Lapkouski et al 2009), the interface of HsdR with the MTase core was not defined when compared with the models presented here. More importantly, the DNA was proposed to bend across the motor domains of HsdR, so that it came near to the endonuclease domain in the same HsdR and could be cleaved.…”

“…It is noteworthy, in this respect, that a process of deassembly of the enzymes occurs after DNA cleavage, and some of the subunits-although not all and depending on the particular type I RM enzyme-can be reused (Roberts et al 2011;Simons and Szczelkun 2011). Lapkouski et al (2009) proposed a more speculative atomic model of EcoR124I using their structure of HsdR, a postulated DNA path across the subunit, and an early, incomplete model of the MTase core (Obarska et al 2006). Although the current models and their model share much in common, there are two main differences; namely, the orientation of the HsdR with respect to the MTase core, and the path taken by the DNA.…”

“…Although the positions of the central MTase core region and the HsdR are well defined, as described above, the orientation of the HsdR in the EM map of both EcoR124I and EcoKI was ambiguous, as the density was rather ring-like in shape. We can, however, propose a model that fits the data and gives the location and directionality of the DNA motor domains by aligning the RecA-like motor domains of the crystal structure of the dsDNA-bound SWI2/SNF2 chromatin remodeling translocase from Sulfolobus solfataricus (Protein Data (Lapkouski et al 2009). As the direction of DNA translocation is defined in the chromatin remodeling translocase, it imposes a similar directionality on each HsdR, and since these have to pull DNA in toward the MTase core of the type I RM enzyme, the orientation of each HsdR relative to the MTase core becomes defined.…”

“…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.…”