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Title: The interactions between EcoKI methyltransferase and specific DNA duplexes
Author: Chen, Angela Her-Ser
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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The S polypeptide of the EcoKI Type I methyltransferase (mtase) has the function of recognising DNA. Specific contacts between EcoKi (mtase) and the DNA containing its recognition sequence (-AAC-(N)6-GTGC-) have been investigated using various substituted oligonucleotides, namely 4-thiothymidine, 5-bromodeoxyuridine, 5-iododeoxyuridine and deoxyuridine. After ultraviolet irradiation, the DNA-S polypeptide crosslinked bands were observed on the specific target recognition DNA, but not on the non-specific DNA by SDS-PAGE gel electrophoresis. The crosslinking of DNA and mtase occurs only between the bottom-strand of the DNA and the S polypeptide as illustrated by labelling the different strands of DNA with γ32P. The crosslinking efficiency between DNA-mtase increased only in one of the nine single sites of BrdU substitution in which the BrdU was substituted at the residue complementary to the first adenine in the -AAC- sequence. This demonstrates a close contact between this sequence and the S subunit at the major groove. Peptide sequencing of various trypsin digested samples of the crosslinked protein-DNA complex further illustrates that the Tyr-27 of the NH2-terminal domain of the S polypeptide is crosslinked with DNA. This is consistent with the role of the N terminal domain of the S subunit in recognizing the -AAC- sequence. By amino acid sequence comparison, Tyr-27 and other small sequence segments can be found at corresponding positions in other two type I enzymes, which have a similar trinucleotide recognition site. In addition the comparison reveals that motif LP-GWEW is partly retained not only in the amino recognition domain but also in the carboxyl recognition domain of the S subunit of type I enzymes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available