February 14, 2007

DNA ends: common tool, different job

Salk News

DNA ends: common tool, different job

La Jolla, CA – Every time a cell repairs or replicates its DNA, the resulting single strand is wrapped up by a dedicated protein complex. In eukaryotes or organisms whose cells have a nucleus, this job is handled by a tripartite complex called replication protein A (RPA). Researchers at the Salk Institute for Biological Studies have now unearthed a novel RPA-like complex that specifically homes in on the short single-stranded DNA “tail” end of yeast chromosomes.

Their findings, reported in the online edition of Nature Structural & Molecular Biology, will help scientists to better understand the dynamics that keep chromosomes ends, called telomeres, intact.

“Identification of this new RPA-like complex, which is targeted to a specific region of the genome, suggests that multiple RPA-like complexes have evolved, each making individual contributions to genomic stability,” says the study’s lead author Vicki Lundblad, Ph.D., a professor in the Molecular Cell Biology Laboratory at the Salk Institute.

With each round of cell division, telomeres – long stretches of repetitive DNA – erode a little bit further. Some have likened this progressive shortening to a genetic biological clock that winds down over time. In fact, when telomeres reach a “critical length,” the cell can no longer multiply – a characteristic sign of cellular senescence.

In certain cells, such as our germ cells or baker’s yeast cells, which need to divide indefinitely, an enzyme called telomerase elongates telomeres to compensate for the continual attrition at chromosome ends. At the same time, telomerase activity is the main mechanism by which human tumor cells achieve immortal growth.

In addition, the natural ends of chromosomes could potentially look like broken strands of DNA that a cell’s repair machinery is designed to fix. This has been a long-standing puzzle, because mending chromosome ends as though they were double-strand breaks would result in either unregulated degradation or end-to-end fusions. Such repair events are lethal for the genome, and in certain settings, can promote the development of cancer.

About 10 years ago, Lundblad discovered that Cdc13, a protein that binds single-stranded telomeric DNA, plays a central role at the telomeres of baker’s yeast. But the function of two Cdc13-associated proteins, Stn1 and Ten1, remained unclear.

When members of the Lundblad laboratory searched the Protein Data Bank for relatives of Stn1, they dug up Rpa2, the middle subunit of the RPA complex. In particular, Stn1 and Rpa2 share similarities in a region known as oligonucleotide/oligosaccharide-binding fold or OB-fold, a protein fold that is commonly used to recognize and bind to either DNA or RNA.

Based on these findings, Lundblad and her colleagues developed a model, which predicts that Cdc13, Stn1 and Ten1 come together at the very end of chromosomes to form a telomere-dedicated RPA-like complex they dubbed the t-RPA complex.

Graduate student and first author Hua Gao tested this model, using detailed biochemical experiments which revealed that Stn1 and Ten1 behave just like their counterparts in the conventional RPA complex. However, Stn1 and Ten1 have the same predilection for single-stranded telomeric DNA as Cdc13, thereby helping to ensure that this complex only targets chromosome tips.

According to Lundblad, this finding raises important questions about the potential biological parallels between the conventional RPA complex and the newly discovered t-RPA complex.

“Whereas the conventional RPA complex acts elsewhere in the genome to raise a red flag to attract the attention of the repair machinery,” says Lundblad, “curiously, the t-RPA complex performs the exact opposite function at chromosome ends, ensuring that these tips do not become targets for DNA repair.”

Lundblad’s group is currently working on elucidating what distinguishes the activities of these two RPA complexes from each other. Researchers who also contributed to this work include postdoctoral researcher Rachel B. Cervantes, Ph.D., and graduate students Edward K. Mandell and Joel. H. Otero.

The Salk Institute for Biological Studies in La Jolla, California, is an independent nonprofit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and the training of future generations of researchers. Jonas Salk, M.D., whose polio vaccine all but eradicated the crippling disease poliomyelitis in 1955, opened the Institute in 1965 with a gift of land from the City of San Diego and the financial support of the March of Dimes.

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