Appointment period: 8/17/2009 to 8/16/2011

Project

Single molecule studies of BRCA2

Objective:

BRCA2 an important protein in homologous recombination responsible for stimulating Rad51 filament formation on single stranded DNA and facilitates Rad51 in displacing RPA from single stranded DNA. BRCA2 is known to bind Rad51, a protein responsible for the homology search and strand exchange during homologous recombination. How BRCA2 interacts with Rad51 and DNA is critical to understand homologous recombination.

Specific Aims:

• Aim 1. Visualize BRCA2 binding to a single strand/double strand DNA substrate during the process of Rad51 loading. Does BRCA2 act at a single strand –double strand junction nucleating Rad51 filament formation? Or does BRCA2 bind randomly to DNA loading Rad51?
• Aim 2. Visualize binding of Rad51 to BRCA2. How many Rad51 proteins are bound to BRCA2?
• Aim 3. Determine the polarity of BRCA2 mediated Rad51 filament formation. Does BRCA2 favor Rad51 filament growth on single stranded DNA over double stranded DNA?
• Aim 4. Determine the oligomeric state of BRCA2 (i.e. monomer, dimer, et cetra). How many BRCA2 proteins are needed for functionality?

Study Design:

Optical traps will be used to hold and manipulate double stranded – single stranded DNA substrates. The DNA will then be introduced to fluorescently labeled proteins such as RPA, Rad51, and BRCA2. Interactions between these proteins and DNA will be directly imaged in real time using fluorescent microscopy.

These experiments will show where BRCA2 binds on DNA, how it stimulates the formation of Rad51 filaments, and how BRCA2 helps Rad51 displace RPA on single stranded DNA. These experiments will also show the polarity of Rad51 filaments while mediated by BRCA2

Atomic force microscope (AFM) will be used to image BRCA2, Rad51 bound BRCA2, and BRCA2 Rad51 interactions. The AFM will provide information on the number of Rad51 proteins bound to BRCA2, the oligomeric state of BRCA2, and information on the polarity of Rad51 filament growth.