Scientists Map Key Moment in Assembly of DNA-Splitting Molecular Machine

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Newswise Stony Brook, NY, October 15, 2014 The proteins that drive DNA replicationthe force behind cellular growth and reproductionare some of the most complex machines on Earth. The multistep replication process involves hundreds of atomic-scale moving parts that rapidly interact and transform. Mapping that dense molecular machinery is one of the most promising and challenging frontiers in medicine and biology.

Now, a team of scientists from Stony Brook University, the U.S. Department of Energys Brookhaven National Laboratory (BNL), Cold Spring Harbor Laboratory, and the Imperial College in London have pinpointed crucial steps in the beginning of the replication process, including surprising structural details about the enzyme that unzips and splits the DNA double helix so the two halves can serve as templates for DNA duplication. Their findings are published today online in the journal Genes and Development.

The genesis of the DNA-unwinding machinery is wonderfully complex and surprising, said study coauthor Huilin Li, a Professor of Biochemistry and Cell Biology at Stony Brook University and a biologist at BNL. Seeing this helicase enzyme prepare to surround and unwind the DNA at the molecular level helps us understand the most fundamental process of life, and how the process might go wrong. Errors in copying DNA are found in certain cancers, and this work could one day help develop new treatment methods that stall or break dangerous runaway machinery.

The research combined electron microscopy, perfectly distilled proteins, and a method of chemical freezing to isolate specific moments at the start of replication. It picks up where two previous studies by Li and colleagues left off. They first determined the structure of the "Origin Recognition Complex" (ORC), a protein that identifies and attaches to specific DNA sites to initiate the entire replication process. The second study revealed how the ORC recruits, cracks open, and installs a crucial ring-shaped protein structure (Mcm2-7) that lies at the core of the helicase enzyme.

But DNA replication is a bi-directional process with two helicases moving in opposite directions. The key question, then, was how does a second helicase core get recruited and loaded onto the DNA in the opposite orientation of the first?

To our surprise, we found an intermediate structure with one ORC binding two rings, said Brookhaven Lab biologist and lead author Jingchuan Sun. This discovery suggests that a single ORC, rather than the commonly believed two-ORC system, loads both helicase rings.

One step further along, the researchers also determined the molecular architecture of the final double-ring structure left behind after the ORC leaves the system, offering a number of key biological insights.

We now have clues to how that double-ring structure stably lingers until the cell enters the DNA-synthesis phase much later on in replication, said study coauthor Christian Speck of Imperial College, London. This study revealed key regulatory principles that explain how the helicase activity is initially suppressed and then becomes reactivated to begin its work splitting the DNA.

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Scientists Map Key Moment in Assembly of DNA-Splitting Molecular Machine