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Structure-Function Differences Among Type I Restriction EndonucleaseAbstractType I restriction and modification systems comprise a three gene operon in which all three genes are required for production of the multifunctional endonuclease; hsdR is absolutely required for restriction, while hsdM and hsdS are required for modification. The hsdS and hsdM genes can produce an independent methylase with a stoichiometry of HsdM2:HsdS1. The HsdS subunit is responsible for DNA recognition while HsdM has been shown to bind the cofactor S-adenosyl methionine (SAM). The cofactor requirements for the endonucleases are SAM, Mg2+ and ATP. The enzymes can function as either restriction endonucleases or as DNA methyltransferases, dependent upon the methylation status of the DNA. For R.EcoKI, SAM is thought to act as an allosteric effector enabling specific DNA binding to occur; while ATP is known to be involved in the determination of the methylation status of the DNA. Unmodified DNA is the natural substrate for DNA cleavage, however the mechanism of cleavage is complicated and results in fragments of random length, with the site of cleavage several thousand basepairs away from the recognition site. Type I restriction and modification systems are divided into three families (type IA e.g. EcoKI, type IB e.g. EcoAI and type IC e.g. EcoR124I) based on gene order, amino acid conservation and enzymatic properties. Within each family there are distinct regions of the HsdS subunit which are strongly conserved. One such region lies about midway between the C- and N-termini and is known as the central conserved region; while the other region is at the C-terminus. Outside of these conserved regions the amino acid sequences are highly variable even between members of the same family and these variable regions appear to be responsible for DNA recognition. We have over-produced the endonuclease of the type IC system EcoR124I in Escherichia coli. The purified endonuclease has a different stoichiometry from that of the type IA R-M system EcoKI. This structural variation is reflected in different cofactor requirements, and related variations in the mechanism by which R.EcoR124I discriminates between hemimethylated and unmethylated DNA. The EcoR124I methylase has been shown to undergo a major structural variation upon DNA binding; while S-adenosyl methionine (SAM) induces a structural change in the EcoKI methylase. The endonucleases, produced by interaction of these methylases with HsdR, might be expected to also show such differences in the mechanism by which DNA binding occurs. |
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