Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577123
Title: Bisubstrate kinetics and processivity measurements on Escherichia coli DNA ligase A
Author: Fraser, Claire Louise
Awarding Body: University of Portsmouth
Current Institution: University of Portsmouth
Date of Award: 2012
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
DNA ligases are essential repair enzymes required for maintaining genomic integrity in cells. The first ligase to be discovered was Escherichia coli DNA ligase; a 670 amino acid, 74 kDa, NAD+ dependent ligase. This work reports a series of studies into the behaviour of His-tagged E.coli ligase. Order-of-addition studies on singly-nicked oligoduplexes under steady state conditions revealed that ligase undergoes an obligatory off-step from the DNA after sealing a break in a phosphodiester strand before readenylation in solution. These results corroborate the findings of Lehman that a sequential model is the normal mode of Ligase operation. Ligase affinity for its substrates NAD+ and DNA were 3.5 μM and 3.5 nM respectively. Length dependency studies on singly-nicked PCR substrates revealed that when two different DNA lengths were in the same solution, the initial association rate was always faster for the longer DNA substrate. For example, 40 bp versus 902 bp gave initial rate values 0.06 nM/min (40 bp) and 0.28 nM/min (902 bp); increasing the length 22 fold increased the initial rate 4 fold. This hints that Ligase uses DNA flanking a nick to locate its specific site. Processivity studies were achieved to determine the one- or three-dimensional pathway of Ligase using doubly-nicked DNA. Nicks were either directly repeated (on the same DNA strand) or inverted (opposite strands). Results revealed Ligase is weakly processive; 32% processive. However, when beta-clamp and gamma-loader were added to the reaction processivity significantly increased.
Supervisor: Gowers, Darren Matthew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Thesis
EThOS ID: uk.bl.ethos.577123  DOI: Not available
Keywords: Biomedical Sciences
Share: