Restriction-Modification Systems
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DNA bindingType I restriction-modification enzymes recognise bipartite sequences, which consist of a 5' specific region of 3-4 bp and a 3' specific region of, usually, 4 bp, separated by a non-specific spacer of 6-8 bp (e.g. EcoR124I recognises GAAn6RTCG - where R = purine and n = any nucleotide). DNA cleavage by Type I R-M enzymes occurs at non-specific sites far from their recognition sequence, which is mediated by ATP-dependent DNA translocation past the enzyme (Yuan et al., 1980; Studier & Banyopadhyay 1888; Dryden et al., 1997; Szczelkun et al., 1997. DNA cleavageType I R-M enzymes are unique amongst all known
restriction endonucleases in that they cleave DNA in a random
manner producing fragments of varying sizes from a few hundred
basepairs to fragments as large as a few thousand basepairs. This random cleavage of DNA by Type I restriction-modification systems is a direct result of translocation of DNA by the HsdR subunit. Translocation of DNA is an ATP-dependent process during which the HsdR subunit hydrolyses ATP and moves the DNA past the enzyme complex, which remains bound at the recognition site. The process is bi-directional, each HsdR subunit acting as a molecular motor, and proceeds until the process is blocked by some external event (usually another enzyme also translocating the DNA; although other blockages include DNA topology). Cleavage follows blockage of the translocation, which will produce a random cleavage site because the process of starting translocation and then blocking translocation is not in any way synchronized. Blockage of DNA translocation can be because of a number of different reasons. As mentioned above the most common reason is collision between two Type I R-M enzymes. This led Studier and Bandyopadhyay (1988) to propose a model for DNA cleavage based upon collision between two translocating enzymes and he was able to clearly show DNA cleavage occurred half-way between two recognition sites on a two-site plasmid. However, our own studies have shown that cleavage of covalently closed circular DNA with a single site is an extremely efficient process, while cleavage of linear single-site plasmid DNA is an inefficient process (Szczelkun et al., 1997). This led us to suggest that DNA cleavage depends upon blockage of the translocation process, which is due to topological barriers to translocation with cccDNA - where translocation can wind all of the plasmid DNA into the expanding loop resulting in stalling due there being no more DNA to translocate. Janscak et al. (1999) have also shown that blockage can also be caused by a Holliday junction. However, many DNA binding enzymes appear to not effect the translocation process and can be displaced from the DNA. Recent studies in our own laboratory have also shown DNA binding drugs and compounds such as ethidium bromide also block translocation. |
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