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Domain conformations of the motor subunit of EcoR124I involved in ATPase activity and dsDNA translocation
BIALEVICH, Vitali
Bacterial type I restriction-modification systems are composed of three different subunits: one HsdS subunit is required for identification of target sequence and anchoring the enzyme complex on DNA; two HsdM subunits in the methyl-transferase complex serve for host genome modification accomplishing a protective function against self-degradation; two HsdR (or motor) subunits house ATP-dependent translocation and consequent cleavage of double stranded DNA activities. The crystal structure of the 120 kDa HsdR subunit of the Type I restriction-modification system EcoR124I in complex with ATP was recently reported. HsdR is organized into four approximately globular structural domains in a nearly square-planar arrangement: the N-terminal endonuclease domain, the RecA-like helicase domains 1 and 2 and the C-terminal helical domain. The near-planar arrangement of globular domains creates prominent grooves between each domain pair. The two helicase-like domains form a canonical helicase cleft in which double-stranded B-form DNA can be accommodated without steric clash. The helical domain, probably involved in complex assembly, exhibits only a few specific interactions with helicase 2 domain. Molecular mechanism of dsDNA translocation, cleavage and ATP hydrolysis has not been yet structurally investigated. Here we propose a translocation cycle of the restriction-modification system EcoR124I based on analysis of available crystal structures of superfamily 2 helicases, strutural modeling and complementary biochemical characterization of mutations introduced in sites potentially inportant for translocation in the HsdR motor subunit. Also a role of the extended region of the helicase motif III in ATPase activity of EcoR124I was probed.
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Interdoménové a intradoménové interakce u motorové podjednotky EcoR124I: Výpočetní studie
SINHA, Dhiraj
EcoR124I is a Type I restrictionmodification (RM) enzyme and as such forms multifunctional pentameric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on the motor subunit HsdR. When non-methylated invading DNA is recognized by the complex, two HsdR endonuclease/motor subunits start to translocate dsDNA without strand separation activity up to thousands base pairs towards the stationary enzyme while consuming ~1 molecule of ATP per base pair advanced. Whenever translocation is stalled the HsdR subunits cleave the dsDNA nonspecifically far from recognition site. The X-ray crystal structure of HsdR of EcoR124I bound to ATP gave a first insight of structural/functional correlation in the HsdR subunit. The four domains within the subunit were found to be in a square planer arrangement. Computational modeling including molecular dynamics in combination with crystallography, point mutations, in vivo and in vitro assays reveals how interactions between these four domains contribute to ATP-dependent DNA translocation, DNA cleavage or inter-domain communication between the translocase and endonuclease activities.
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