July 30, 2015

Study sheds light on crucial DNA binding protein

Researchers at Vanderbilt University have established the molecular basis for the function of Replication Protein A (RPA), a DNA binding protein that is a crucial “scaffold” for genome replication, response to damage and repair.

Researchers at Vanderbilt University have established the molecular basis for the function of Replication Protein A (RPA), a DNA binding protein that is a crucial “scaffold” for genome replication, response to damage and repair.

With colleagues at Yale University, they also determined that the small, highly charged protein DSS1 acts on RPA to function as an essential co-factor for the key tumor suppressor BRCA2 (breast cancer susceptibility protein 2). Individuals with BRCA2 mutations exhibit genomic instability and are predisposed to breast, ovarian and other cancers.

In the journal Molecular Cell, the researchers report that DSS1 functions as a DNA “mimic” for RPA. DSS1 “remodels” RPA so it releases its strong grip on DNA to enable processing by BRCA2 and repair of devastating DNA double-strand breaks.

Walter Chazin, Ph.D.

However, DSS1 is elevated in tumor samples, and higher DSS1 expression is associated with therapy resistance and poor prognosis. Inhibition of DSS1 expression in these tumors, by alleviating resistance, could enhance standard chemotherapy, the researchers conclude.

The DSS1 studies were led by Walter Chazin, Ph.D., Chancellor’s Professor of Medicine, professor of Biochemistry and director of the Vanderbilt Center for Structural Biology, and  Patrick Sung, D.Phil., Ph.D., professor of Molecular Biophysics and Biochemistry and of Therapeutic Radiology at Yale.

Claudia Wiese, Ph.D., assistant professor of Radiation Cancer Biology at Colorado State University, was co-senior author of the Molecular Cell paper.

“This study resolves a long-standing conundrum about how DNA gets transferred from RPA to BRCA2,” said Chazin.

Sung added, “Our collaborative, multidisciplinary approach was essential for understanding the very high degree of biological and mechanistic complexity of DSS1 action at the critical initial stage of the repair of highly toxic DNA double stand breaks.”

The importance of RPA in maintaining and propagating the genome is due to its ability to bind very strongly to single stranded DNA (ssDNA). Since DNA is so important, it is stored with a complete back-up copy, and the two copies together make up the well-known DNA double helix.

This helical storage form of DNA is very stable, but in order to read the DNA code, it is necessary to unwind the double helix into its separate strands. Unwound single strands of DNA (ssDNA) are very susceptible to being damaged and readily form irregular tangles.

Nature therefore evolved proteins such as the RPA protein in humans to protect ssDNA and keep it organized. RPA is a very complicated protein and it has remained challenging to understand how it functions.

An important step forward was made when the Chazin group reported last month a description of the functional dynamics of RPA in the journal Structure. Chazin and his colleagues used NMR (nuclear magnetic resonance) techniques to define how RPA domains, portions of the protein with distinct biochemical functions, move in space.

Their work demonstrated how RPA is able to use some of its eight domains to bind to sections of DNA of interest, and simultaneously use other domains to recruit the proteins required to replicate or repair this DNA.

In a commentary written in the same journal, Patrick Loria, Ph.D., professor of Chemistry and of Molecular Biophysics & Biochemistry at Yale, noted “In this elegant work, Chazin and co-workers show in part how the flexibility of RPA enables such a complex set of interactions to occur.”

The two studies add to the growing body of data detailing the intricacies of DNA replication and repair, and suggest it may be possible to modify the function (or compensate for the dysfunction) of various essential proteins, including BRCA2, RPA and DSS1, to stop tumor growth and improve therapeutic outcomes.

The study in Structure was supported in part by National Institutes of Health grants GM065484 and CA092584. The study in Molecular Cell was supported in part by NIH grants ES0125252, ES007061, CA168635, CA092584 and ES021454.