Structure of the motor subunit and translocation model for EcoR124I restriction-modification complex

Rüdiger Ettrich¹, Mikalai Lapkouski¹, Santosh Panjikar², Pavel Janscak³, Ivana Kuta Smatanova¹, Jannette Carey^4*, Eva Csefalvay¹

¹Department of Structure and Function of Proteins, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic; and Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic

²EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D22603 Hamburg, Germany

³Institute of Molecular Cancer Research, University of Zürich, Wintherthurerstrasse 190, CH-8057 Zürich, Switzerland

⁴Chemistry Department, Princeton University, Princeton, New Jersey 08544-1009, USA

Ettrich@greentech.cz

Keywords: X-ray crystallography, Protein-Nucleic Acid Interactions, Protein Function

Introduction

Although type I restriction-modification systems of bacteria were the first to be discovered and characterized, their lack of specific cleavage sites relegated them to the sidelines of early DNA enzymology while the site-specific type II systems were developed into the essential reagents that today make molecular cloning routine. Nevertheless, the intriguing complexities of type I mechanisms led to an extensive body of sometimes puzzling results, in sharp contrast to the straightforward mechanisms of the type II restriction-modification systems (RMs). Type I RMs are multisubunit, multifunctional molecular machines that recognize specific, typically asymmetric, DNA target sequences of ~13 to 17 bp. Depending on the methylation status of adenine residues in the target, three enzyme subunits either act together as a typical methyltransferase or recruit a pair of endonuclease motor subunits that initiate translocation of DNA through the enzyme and eventually cleave non-specifically at apparently random sites. The protein complex remains bound at the target sequence while up to thousands of bp are pumped through the enzyme by tracking along the helical pitch at rates of up to hundreds of bp per second. Translocation is driven by helicase-like motor subunits that consume ~1 ATP per ~1 bp without separating the strands.

Results and Discussion

The type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes act as conventional adenine methylases at their specific target sequences, but unmethylated targetsinduce them to pull thousands of basepairs through the enzyme beforecleaving distant sites nonspecifically. Biochemical, biophysical, and molecular biological studies of their translocation and cleavage mechanisms offer a wealth of detail that has lacked a structural framework. We report the first x-ray crystal structure of the subunit responsible for DNA translocation and cleavage by the type I enzyme EcoR124I, resolved at 2.6 A [1]. Understanding how the cooperation of subunits, domains, substrates, and cofactors enables type I RMs to carry out their diverse and peculiar activities is likely to be enhanced by knowledge of their molecular structures. The crystal structure reported here of the HsdR motor subunit of plasmid-borne type I RM EcoR124I is used to develop a model for the complete translocation complex with bound DNA, using structures of related methylase and specificity subunits and constraints from experimental data on the pentameric enzyme complex on DNA. The model predicts the rearrangements and cooperation of subunits and domains required to initiate and stabilize the translocating complex as it tracks on DNA. The model accounts for many known features of type I RMs, and makes a number of experimentally testable predictions about their structural and functional organization and mechanism and provides a structural framework for duplex DNA translocation by RecA-like ATPase motors.

Reference

1. Mikalai Lapkouski , Santosh Panjikar , Pavel Janscak , Ivana Kuta Smatanova , Rudiger Ettrich , Eva Csefalvay, Structure of the motor subunit and translocation model for EcoR124I restriction-modification complex, Nature Structural & Molecular Biology, 2009 Jan;16(1):94-5. Epub 2008 Dec 14. doi: 10.1038/nsmb.1523

Acknowledgements.

We gratefully acknowledge support from Ministry of Education, Youth and Sports of the Czech Republic [MSM6007665808, LC06010]; Academy of Sciences of the Czech Republic [AVOZ60870520]; Grant Agency of the Czech Republic [203/08/0114 to R.E].