Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes

Abstract

The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5′-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can ‘turnover’ in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase–nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

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

“…One may speculate that this great flexibility would allow the RM enzyme to accommodate the stresses built up during the extensive DNA translocation periods observed for these molecular machines. 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).…”

Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

“…Furthermore, the anaerobically essential gene Dshi_3758 encodes a transposase of an IS4 element. The gene Dshi_2313 encodes an HsdR family type I DNase as part of a restriction-modification system (43). In Mycoplasma, the HsdSMR enzyme system has been shown to be activated by high-frequency gene rearrangements (44) and the whole system is controlled by proteolysis (45).…”

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

“…5 nM Triplex was pre-incubated with 40 nM MTase and 120 nM wt or mutant HsdR at RT °C for 5 min. Following Simons & Szczelkun (2011) the required concentration of the HsdR subunit for R 2 M 2 S 1 complex formation is twice that of the DNA under the given conditions. A 120 nM concentration of HsdR (methyltransferase (M 2 S 1 ) to HsdR ratio 1:3) was enough to saturate both methyltransferase binding sites under experimental conditions.…”

“…Excess of DNA concentration over enzyme was used (6:1 ratio) following Janscak, Abadjieva & Firman (1996) and 6-folds excess in HsdR subunit concentration ensures efficient ATPase activity as was described by Janscak, Dryden & Firman (1998). The HsdR subunit does not turn over after cleavage of supercoiled DNA that might occur, nevertheless, ATPase activity is efficient under tested conditions (Simons & Szczelkun, 2011). Reactions (40 µL) were started by addition of ATP mixture containing 0.16 µCi (0.0013 mM) [ γ 32 P]-ATP to a final concentration of 2 mM and incubated at 37°C.…”