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Spaghetti on a fork

Reconstructing proteins in the lab is a challenging task

Imagine you’ve entered a contest to cook the world’s biggest ball of spaghetti. You have to meet the following criteria: the ball has to be made of a single spaghetti strand; the strand has to be entwined in a very exact way; and you have to be able to pick up the ball without the entire thing falling apart.

It may sound odd but cancer researchers have been struggling with a similar problem for years – one that has just been solved.

The cause of the problem is a protein called BRCA2, which plays a vital role in protecting people against cancer. BRCA2 is one of the largest of all human proteins, and it’s a bit like a miniature version of our giant imaginary spaghetti ball: it’s a single, long chain of amino acids entwined in a very precise way.

Because of its enormous size, it’s very hard to produce in the lab, which makes things very difficult for cancer researchers who want to understand how it works.

Until recently, scientists have had to make do with studying small fragments of the whole protein, or smaller versions taken from other living things like tiny worms.

But no more: three independent teams, including Cancer Research UK’s Professor Steve West, have finally managed to purify the hefty protein, publishing their impressive technical feat in the journal Nature Structural and Molecular Biology this week.

BRCA2 and DNA damage

Inheriting a faulty copy of the BRCA2 gene (which contains instructions telling our cells how to make the colossal BRCA2 protein) significantly increases a woman’s chances of developing breast cancer. Around 2 to 5 per cent of breast cancer cases are thought to be due to faulty BRCA genes, and they also play a role in a small number of ovarian and prostate cancers.

The discovery of the BRCA2 gene in the 90s – helped by funding from Cancer Research UK – led to genetic tests, and even drugs specifically designed to target BRCA faults.

But despite all these advances, it’s surprising to discover that researchers understand relatively little about what BRCA2 actually does inside our cells – and why faults in the gene, or the protein it produces, should have quite such potent effects.

So far, researchers have known that BRCA2 is involved in helping cells to repair damage to their DNA – the genetic ‘instruction manual’. Cells lacking the gene quickly accumulate DNA mistakes as they grow and multiply, damaging these vital instructions and causing them to lose control of how often and where they multiply, ultimately leading to cancer.

What wasn’t clear was exactly how BRCA2 was involved in the repair process. But answering this question in detail couldn’t be done without actually isolating the protein for study.

As their new paper describes, some modern molecular biology tricks enabled Professor Steve West and his colleagues in the UK, US and Denmark to finally crack the problem and purify samples of the human BRCA2 protein.  At the same time, this impressive feat was managed in slightly different ways by two other labs in the US.

But what does it do?

Having purified human BRCA2, the scientists then put it through its paces in the lab, finding out exactly how it was involved in helping cells to repair DNA damage. In one set of experiments, they used electron microscopy to spy on how BRCA2 interacts with DNA.

Impressive images (see below) show individual BRCA2 proteins attached to rope-like lengths of DNA, just a few hundredths of a millimetre wide.

BRCA2 binding to DNA

Using an electron microscope, the scientists could see blobs of BRCA2 protein attached to strands of DNA

Combined with the results of other tests, the data shows that the BRCA2 protein homes in on the broken ends of DNA strands. Broken DNA tends to unravel, resulting in a bad case of split ends. BRCA2 recognises and latches onto these split ends.

But BRCA2 doesn’t work alone – it gets a hand from another crucial DNA repair protein called RAD51. Summoned by BRCA2, RAD51 helps to patch together the split ends. It does all the heavy lifting but BRCA2 tells it where to go.

Professor West explains:

“This important step allowed us to discover how the BRCA2 protein helps to repair DNA breaks – that can ultimately lead to breast cancer – by guiding DNA repair proteins to help fix the damage.

“Understanding how BRCA2 masterminds the DNA repair kit to prevent tumour formation is a vital step towards developing new treatments for this type of breast cancer.”

This discovery is not going to immediately change the way that people with BRCA2-related cancer are treated. But it’s an extremely important finding for cancer and DNA repair researchers around the world, as they work to understand the disease and develop more effective treatments.

As we’ve written about before, half the battle in cancer research is developing the tools and techniques to answer vital questions. And now BRCA2 has yielded up some of its secrets, we can hope to see much more progress made in this area in the future.

Kat and Ed

References

Thorslund, T. et al (2010). The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA Nature Structural & Molecular Biology DOI: 10.1038/nsmb.1905

Jensen, R., Carreira, A., & Kowalczykowski, S. (2010). Purified human BRCA2 stimulates RAD51-mediated recombination Nature DOI: 10.1038/nature09399

Liu, J., Doty, T., Gibson, B., & Heyer, W. (2010). Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA Nature Structural & Molecular Biology DOI: 10.1038/nsmb.1904