Download DNA REPAIR AND CANCER: ROLE OF THE BREAST

DNA REPAIR AND CANCER: ROLE OF THE BREAST CANCER SUSCEPTIBILITY
PROTEIN BRCA2
Oscar A. Llorca
Institute of Cancer Research
237 Fulham Road, London
Oscar Llorca, Janet Valentine, Alan Ashworth and Keith R. Willison
1- The Brca2 protein
Around 10% of all breast cancers have a familiar incidence. Of those, around 50% are associated
with mutations in one or two of the breast cancer susceptibility genes, BRCA1 and BRCA2.
These two genes codify two proteins, which are not related in sequence, but share as a common
property that they are big proteins (200 and 400 Kda, respectively), which are able to interact with
many different partners to form large macromolecular complexes.
Of particular interest, Brca2 contains eight BRC repeats, a conserved motif of about 30-40
residues, involved in Rad51 binding. Rad51 is the most essential component of the machinery
required for DNA double strand breaks repair, through the process known as homologous
recombination.
The exact function of the Brca2 protein is unknown, as it is its role in breast cancer development,
but current models suggest that Brca2 might regulate the assembly of macromolecular complexes
require repairing the DNA breaks. Brca2 might work as a tightly regulated scaffolding protein.
2- Electron microscopy and 3-D reconstruction of macromolecular complexes
In the past decade it has become clear that knowing the structure of a protein provides with
essential information about how the protein works and carries out its duties. X-ray crystallography
is the tool of choice for high-resolution studies. Nevertheless, big proteins are more difficult to
crystallise. So, these studies tend to be restricted to the analysis of smaller domains within the big
protein.
The structural studies of large macromolecular assemblies where different proteins interact are
even more complicated, as usually those complexes are extremely difficult to purify in the
quantities needed in high-resolution techniques.
The electron microscopy of single particles and 3-dimensional reconstruction techniques can
provide a medium resolution map of the complete macromolecular complex under physiological
conditions using low amounts of material. The information provided can help to understand the
protein function in the framework of its whole three-dimensional shape.
Both Brca1 and Brca2 proteins are large polypeptides taking part of even larger macromolecular
complexes. This fact, together with the difficulties in purifying the proteins in large amounts, has
made difficult the structural studies of both breast cancer susceptibility proteins.
My goal in this project is to be able to purify the brca2 monomer and its complexes to carry
electron microscopy analysis and three-dimensional reconstructions.
3- Purification of Brca2 complexes
We have derived ES cells carrying an endogenous BRCA2 gene C-terminally tagged with myc
epitopes. We have shown that these tagged proteins are apparently functional in DNA repair. I
have used sucrose gradient fractionation to resolve different Brca2 complexes according to their
size. These complexes were used in anti-myc antibody purification. This material is being used
for preliminary electron microscopy studies, but the final yields are low, making difficult any
further analysis. Hence, we have started to generate a new allele of Brca2 in S cells, in which the
gene is tagged with the TAP (Tandem Affinity Purification) tag. This system utilises a dual tag.
Protein complexes are bound to an immuno-globulin column via a C-terminal protein A domain.
This system has been specifically designed for the purification of low abundant macromolecular
complexes under physiological expression levels.
We will use a combination of sucrose gradients and the double tag affinity purification to obtain
enriched Brca2 monomer and Brca2 complexes. We expect to get enough material to carry out the
electron microscopy and the three dimensional reconstruction of the different complexes.