Damage Control

Damage  ControlBroken DNA—be it a gap, a nick or a double strand break —is a hallmark of cancerous or aging cells. But once key kinases and their regulators detect DNA damage, a molecular cascade swings into action, triggering posttranscriptional changes in proteins that eventually spur enzymes, such as ligases, to repair the damage and facilitate further replication. Until recently, little was known about exactly how mammalian cells sense DNA damage and coordinate these responses and which molecules act to set things right.

By the mid-1990s, researchers had explored the DNA damage response (DDR) using simple model organisms, such as yeast, and found that DDR was a signal transduction pathway with two kinases, namely ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related), orchestrating a suite of phosphorylation events. About 100 proteins were implicated in the response, and about 25 of those were known to be actual kinase phosphorylation substrates.

Then Stephen Elledge, Steven Gygi and a team of researchers from Harvard Medical School irradiated cells with ionizing particles to trigger DNA damage. The team calculated the rate of phosphorylation in irradiated versus undamaged human embryonic kidney cells by immunoprecipitating phosphorylated peptides with attached phospho-antibodies from two cell cultures: One grown in the presence of isotopic (or heavy) amino acids. Using mass spectrometry to sort out light and heavy peptides, the researchers found that phosphorylations increased more than fourfold after irradiation and uncovered more than 700 proteins that were activated either directly or indirectly by ATM and ATR in response to DNA damage. Their results, as described in this month’s Hot Paper, were published in a May 2007 issue of Science. 1

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