Single -Molecule and Ensemble Fluorescence Studies of Single -Stranded DNA and Replication Proteins

DNA replication is a complex process involving many proteins that must be performed for a cell to proliferate. In this work, we will study three members of the replication system: single-stranded DNA, single-stranded DNA binding proteins, and the sliding clamp and clamp loader proteins. Fluorescence studies have been widely used to study the properties of biomolecules and here we will use both Fluorescence Polarization Anisotropy (FPA) and Fluorescence Resonance Energy Transfer (FRET) to study these biomolecules. First, we present a method by which single molecule FRET measurements can be used to determine the persistence length of single-stranded DNA. Our results indicate that the persistence length decreases from 3.0nm to 1.5nm as the salt concentration increases from 50mM NaCl to 2M NaCl. Furthermore, we use fluoresce anisotropy and FRET measurements in ensemble studies to examine properties of single stranded DNA binding proteins from archaeal organisms. Our results will show that the Archaea have exploited the single stranded DNA binding fold motif unlike the other domains of life to create a wide variety of proteins that have different binding properties leading to the probable conclusion that many of the proteins area a result of gene duplication and recombination events. Finally, we will examine the properties of the clamp loader protein as it loads a fluorescently labeled clamp to DNA on both the ensemble and single molecule measurements. Our findings show that loading the clamp is a multi-step process with different conformational states that can be observed for the first time at a single molecule level. We also will examine mutations of the clamp loader protein to bring further insight into the not fully understood Methanosarcina acetivorans clamp loader.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics and Computational Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• McKinney, Mary Cathleen

Contributors dc:contributor

• Ha, Taekjip

• Biophysics, General

Language dc:language

eng

Identifier

(MiAaPQ)AAI3242936

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/85456