Abstract
The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5′-GCCGC-3′ site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes.
Highlights
Restriction-modification (RM) enzymes are important player in protecting bacterial cells against invasion by foreign DNA [1,2,3]
On the basis of the DNA cleavage and triplex displacement experiments, we propose that CglI translocates bidirectionally along the 1D DNA contour from its target site and cleaves dsDNA when two R2H2·CglI complexes collide with each other
We investigated the mechanisms of DNA hydrolysis and long-range communication of the CglI restriction enzyme
Summary
Restriction-modification (RM) enzymes are important player in protecting bacterial cells against invasion by foreign DNA [1,2,3]. Examination of the communication mechanism and of the effect of different arrangements of targets on DNA cleavage activity can distinguish between the different mechanisms: i) For the classical Type I RM enzymes, there are two independent motors subunits (HsdR) that catalyze stepwise translocation along the DNA up- and downstream from the site; i.e. translocation is simultaneously bidirectional (Figure 1A) [10,11,12]. DNA cleavage occurs at random non-specific sites when two motors collide along the 1D DNA contour (forming a ‘collision complex’). On a plasmid this can be achieved by two motors from the same complex and so a single site is sufficient to produce cleavage. The Type I target sites are asymmetric but since translocation is bidirectional, there is absolutely no effect of the relative orientation of pairs of sites on
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