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Researchers identify mechanism of cancer caused by loss of BRCA1 and BRCA2 gene function
Date:4/28/2014

other diseases.

"Some years ago, we and others suggested that BRCA1 and BRCA2 regulate homologous recombination at sites of stalled replication," explains Scully. "We believe that this function is critical to how these genes suppress breast and ovarian cancer. Until now, we haven't had the tools necessary to study in molecular detail the HR processes at sites of replication fork stalling in the chromosomes of a living mammalian cell."

To solve this problem, first author Nicholas Willis, PhD, a postdoctoral fellow in the Scully laboratory, created a new tool by harnessing a protein-DNA complex that evolved in bacteria.

"We found that the Escherichia coli (E. coli) Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR in mouse cells," explains Willis. "In its essence, E coli bacteria a standard model organism in science has evolved a very simple system to arrest replication forks in a site-specific manner." This system, he explains, is composed of short DNA elements called Ter sites, 21-23 base pairs in length and tightly bound by the protein Tus. "Tus binds these Ter elements with extremely high affinity and, upon replication fork approach, acts as a barrier to fork progression along the DNA. Tus/Ter effectively sets up a 'road block' and stalls the replication fork."

The acid test for the new tool, say the authors, came when this same short Ter sequence was placed into a reporter, a slightly larger DNA sequence that can undergo certain rearrangements within a chromosome when triggered to do so. "When it engaged in homologous recombination, a change in the sequence caused the cells to express green fluorescent protein," explains Willis. "When the cells glowed green, we knew we had a positive event."

As he goes on to explain, the team adapted the reporter to distinguish between error-free/high-fidelity HR and an error-prone/aberrant form of HR. "Remarkabl
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Contact: Bonnie Prescott
bprescot@bidcm.harvard.edu
617-667-7306
Beth Israel Deaconess Medical Center
Source:Eurekalert

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