Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins

RAD51 protein plays an important role in homologous genetic recombination (HR), an essential DNA metabolic process used by cells to faithfully repair the most deleterious forms of DNA damage and maintain genomic integrity. RAD51 along with its bacterial counterpart RecA, bacteriophage UvsX and archaeal RadA have been subjected to genetic and biochemical scrutiny resulting in a plentitude of mechanistic and functional information on formation, regulation and activities of these recombinases. An important disconnect between these two lines of investigation still exists because the recombinase functions of RAD51 are highly regulated through mediator proteins like the BRCA2 recombination mediator, and a host of post translational modifications, namely phosphorylation. The mechanism and biochemical implications of these regulatory processes have not been satisfactorily evaluated in-vitro. This work characterizes the interaction between RAD51 and the BRCA2 recombination mediator protein using computational methods to generate homology models for this interaction which are validated through experimental data. Using the knowledge gained from our structural model for the RAD51 recombinase, I developed a novel strategy to understand several key mechanisms for the regulation of RAD51 by phosphorylation. RAD51 is phosphorylated by the cABL tyrosine kinase. The mechanistic and functional significance of this event is largely disputed. Using biochemical and single molecule assays reconstituting major activities of RAD51, I have successfully dissected the biochemical mechanism of regulation of RAD51 by the c-Abl kinase. The results of this work strongly correlate with observations made in previous cell based analysis.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2016

Author dc:creator

• Subramanyam, Shyamal

Contributors dc:contributor

• Spies, Maria
• Schuler, Mary A.
• Tajkhorshid, Emad
• Gennis, Robert B.

• RAD51 Recombinase
• DNA Repair
• DNA Damage
• Protein-Protein Interactions
• Post Translational Modifications
• Phosphorylation
• Recombination Mediator
• c-Abl Kinase
• BRCA2
• Single Molecule Total Internal Reflection Microscopy
• Homology Modeling

Statement dc:rights

• Copyright 2016 by Shyamal Subramanyam

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/90879

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/90879