Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Abstract

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

“…Here we describe experiments on the multidomain and multifunctional s ingle p olypeptide Type I SP restriction endonucleases, as exemplified by LlaGI and LlaBIII from Lactococcus lactis ( 6 , 7 ). Rather than being able to directly dimerise, structural and biochemical studies predict that Type ISP nuclease monomers are held at least 30 bp apart ( 8 ). To produce a dsDNA break it was suggested that the enzyme complex moves back-and-forth on the DNA while introducing multiple nicks, eventually shredding the DNA.…”

“…To avoid this, Type ISP endonucleases control their nuclease activity using long-range communication. Where there are a pair of unmodified targets in a head-to-head (HtH) orientation on foreign DNA, dsDNA breaks are produced at random, non-specific locations, with the majority between the sites ( 8 – 11 ). However, for every pair of HtH sites on newly-replicated host DNA, one target will retain methylation from the parental strand.…”

“…Where the pair of enzymes initiate from sites in HtH orientation, the translocation is convergent and the collision complex is described as ‘head-on’. In this arrangement, each strand is cut exclusively by just one of the enzymes to give 3′ overhangs ( 8 , 11 , 13 ). Where the pair of enzymes initiate from sites in HtT orientation, collision complexes would be described as ‘rear-end’ (see below).…”

“…DNA contacts/cleavage are defined by data from Chand et al . ( 8 ). DNA numbering is based on the methylated adenine (+1), with positive values in the downstream direction of translocation.…”

“…The yellow box represents the minimum region contacted by the nuclease. The N-terminal nuclease domain contacts the DNA at approximately -30 bp (as judged by an ATP-independent nicking event) ( 8 ). Downstream translocation occurs in a rightward direction in this view.…”