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It has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system. But, when target sequences within the chromosome of one such bacterium (Escherichia coli K-12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated . Thus, the resident chromosome is recognized as ‘self’ rather than ‘foreign’ even in the absence of DNA modification. Makovets et al (2004) now provide evidence that restriction alleviation may be a characteristic of other Type I restriction–modification systems, and that it can be achieved by different mechanisms. Their experiments support disassembly of active endonuclease complexes as a potential mechanism. Makovets et al, identified amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrated that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment. In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome. ClpXP proteolysis of HsdR occurs only when the HsdR subunit is a part of a functional endonuclease holoenzyme, which is able to translocate and cleave DNA, as shown by a mutational analysis where mutations that prevent ATP-dependent DNA-binding at the recognition sequence and subsequent DNA translocation also prevent the vulnerability of the HsdR subunit to ClpXP degradation (Doronina and Murray, 2001). In contrast, mutations that simply block restriction activity (e.g. Motif X mutants) do not effect the HsdR susceptibility to ClpXP proteolysis. Doronina and Murray also indicated that under conditions of Restriction Alleviation the bacteria maintained low-level restriction activity (against incoming phage), which appeared to be localised within the cell "possibly in association with the membrane". This restriction activity maybe associated with EcoKI molecules that have been shown to be membrane associated Holubova et al., 2000, 2004) through HsdR (which is also likely to afford HsdR with protection against ClpXP proteolysis). Therefore, this residual restriction activity appears to be effective against incoming 'foreign' DNA, but is unable to cleave unmodified chromosomal DNA produced during conditions that induce Restriction Alleviation. A simple conclusion from these observations is that conformational changes associated with initiation of translocation induce ClpXP susceptibility within HsdR. However, Doronina and Murray showed that ATP hydrolysis (i.e. DNA translocation itself) was a prerequisite of ClpXP proteolysis. |
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