Life is possible thanks to the process of DNA replication. It is a feat almost every cell in the human body undergoes. Yet, until fairly recently, the exact mechanism for DNA replication remained a mystery.
To better understand DNA replication, Huilin Li and his colleagues at Stony Brook, along with Brookhaven scientists, teamed up with researchers from Cold Spring Harbor Laboratory and Imperial College in London. The group’s latest work is part of series of studies published in the journal Genes and Development last month that has begun to uncover the protein machinery responsible for DNA replication.
“When unwound, all the DNA in a human cell measures two meters long,” Bruce Stillman, co-author as well as president and CEO of Cold Spring Harbor Laboratory, said. “This has to be copied in about eight hours, at a rate of about 60 base pairs per second. Not surprisingly, DNA replication is one of the most highly controlled processes in a cell.”
When the copying is imperfect, mutations can arise in the DNA of a cell. If the resulting mutation is severe, large-scale deletions or duplications may occur. In some cases, these give rise to tumor cells that spread to cause cancer. Or, if the mutations occur during the production of sperm or eggs, disorders such as autism can arise in children.
While the cancerous effects of changes in DNA have long been known, understanding the way mutations (and normal DNA) are replicated would require scientists to use a purified cell-free system.
“In the mid-eighties, we realized that using yeast would combine both the biochemical and genetic approaches,” Stillman said. “Because of high conservation, what is true for yeast is generally true for humans. Though some of the details are different, we study both and can flip back and forth between them.”
Stillman helped pioneer one of the first cell-free DNA replication systems, for which he was awarded the 2010 Louisa Gross Horwitz Prize for Biology or Biochemistry.
Perhaps unknown to some, the early history of replication was intimately tied to Stony Brook’s own history.
“It largely started with York Marahrens, a Stony Brook graduate student in the late 1980s, who published the first detailed analysis of the origin of replication in the budding yeast,” Stillman said. “A short time after, Steve Bell, then a postdoc in my lab, identified a protein that binds to the origin of replication, which was named ORC.”
This initiator protein ORC, or origin replication complex, first binds to the DNA. It then recruits several other proteins in a stepwise fashion that, together, unzip and prime the DNA for replication. Yet with so many tightly-regulated components, the exact mechanism has taken years to dissect, piece by piece.
In their latest study the group looked at the structure of the helicase, a ring-like protein responsible for unwinding the double-stranded DNA into two single strands.
“If you think about it, DNA has to somehow get through the middle of the helicase, which is shaped like a barrel,” Stillman said. “The replication machinery must open up the barrel, load the helicase onto DNA and then activate it. Somewhat surprisingly, it turns out this mechanism is related to a previous mechanism we studied for loading other DNA replication proteins onto DNA.”
With a better understanding of the helicase structure, scientists have started to get an idea about how the helicase is loaded and how it functions once part of the replication machinery. Yet, many pieces still remain to be understood.
“Once the helicase is loaded, it has to be activated to begin unwinding the DNA,” Stillman said. “This activation process is one of the areas we are focusing on. In particular, it seems activation has to be coordinated over the entire genome.”
Because DNA replication is such a fundamental process in cells, a more complete model may yield new insights that have far-reaching implications. Everything from development to cell death to cancer seems to somehow link back to DNA replication. Once researchers understand how this process works, they may find entirely new ways to regulate it, even stopping it when it goes awry.