| Popis: |
Production of polyamines and nucleoid-associated proteins is tightly regulated and restructures the nucleoid-associated proteins is tightly regulated and restructure the nucleoid under environmental conditions that induce DNA damage into an even more highly condensed conformation. These ‘stressful’ conditions can cause the specific methylation sequence of DNA to be lost, which leaves the DNA open to self-attack by restriction enzymes. One such enzyme is EcoKI, a type I restriction enzyme that protects the bacterial cell by destroying foreign invading DNA. Upon loss of specific methylation, EcoKI could potentially destroy the host DNA and kill the bacteria. This damaging restriction is alleviated due to partial proteolysis of EcoKI by C1pXP, although a reduced ability to destroy incoming foreign DNA is maintained. However, this method of alleviation does not exist for all type I enzymes, implying that additional restriction alleviation is required to protect bacteria. In this thesis, it has been found that the condensed structure of DNA produced by the polyamine spermidine and the nucleoid-associated protein StpA, as well as the non-specific DNA-binding of the ligand YOYO, dramatically inhibit EcoKI ATP hydrolysis an restriction activities. These results show that condensation may be a method used by bacteria to protect the nucleoid from self-attack by EcoKI under DNA-damaging conditions, and therefore forms a second mechanism of restriction alleviation. Such a condensed DNA structure may inhibit access of the enzyme to its binding site as well as inhibiting the physical ability to translocate DNA. This is in contrast to invading foreign ‘naked’ DNA in the cytoplasm, which adopts a more open conformation, and therefore forms an ideal substrate for EcoKI translocation and restriction. |
