Walter Chazin, Ph.D., and Ellen Fanning, Ph.D., discuss data related to their recently published model of DNA replication. The model resulted from a successful interdisciplinary effort between the two labs.
photo by Anne Rayner
VUMC research team piece together DNA replication in ‘Nature’
Although DNA replication is one of the most fundamental, defining characteristics of life, the enigmatic molecular details of the process have eluded scientists for decades. By studying DNA replication in simian virus 40 (SV40), a virus that infects monkeys, an interdisciplinary team of Vanderbilt scientists recently demystified part of this process.
The team, led by Walter Chazin, Ph.D., professor of Biochemistry and Physics, and Ellen Fanning, Ph.D., Stevenson Professor of Molecular Biology, reported their results in the April edition of Nature Structural and Molecular Biology.
Although primarily a monkey virus, since SV40 can replicate its DNA in human cell extracts, it is a good model for studying DNA replication. The virus carries its genetic information on a circular piece of double stranded DNA. Within this hula-hoop shaped piece of DNA lays a single point at which DNA replication begins, appropriately called the rigin.' At this origin, a team of proteins converges to unwind the DNA double helix and present a naked single strand of DNA (ssDNA) to the proteins that copy its sequence.
“We already knew the factors (proteins) involved in DNA replication in SV40,” Fanning said. “We could identify a subset of those factors that was sufficient to initiate replication, only four proteins, and we knew that those proteins interacted with each other.”
Of those four critical proteins, the simple SV40 genome encodes for only one, called the large T antigen (Tag). But the cunning viruses mooch off of the cells they invade, co-opting the other three required proteins from the host's DNA processing machinery to begin copying the viral genome.
The sole viral protein, Tag, unwinds the double-stranded DNA and ushers in a host enzyme, called DNA polymerase-primase (pol-prim), that copies the naked DNA. Another necessary host protein, the human replication protein A (hRPA), binds to the ssDNA, protecting it from enzymes that might degrade it and keeping it straight and untangled so that pol-prim can begin copying it.
“We knew that Tag and hRPA had to interact to initiate replication, but we wanted to know 'why' and 'how,'” Fanning explained.
About four years ago, Chazin, a structural biologist and director of the Vanderbilt Center for Structural Biology, and Fanning, a molecular biologist/biochemist, brought together by a shared interest in DNA replication, merged their divergent but complementary talents to address those questions.
To see how and where Tag and hRPA interact, the researchers snapped detailed pictures of the two proteins interlocked in a molecular embrace.
“We had structures of the two proteins in isolation,” Chazin said. “So we mapped in detail the surfaces where they interact and generated a 3-D structure of the complex.”
These molecular snapshots showed that a specific piece, or domain, of the human protein, hRPA32C, and Tag physically touch at multiple points.
“Once we had that information, we could determine exactly which (amino acid) residues were critical,” Chazin said. “That suggested mutations we could make. Then we characterized those mutations biophysically.”
They found that mutations at specific points in each protein reduced their attraction to each other. When placed in the context of the 'real' or native hRPA protein, they could then uncover the importance of those residues.
Chazin and Fanning determined that, even with the mutations, the Tag-hRPA complex could bind to the origin and unwind the double stranded DNA, but it did not allow the synthesis of primers (short segments of RNA that are formed at the very beginning of replication).
“That's a good hint for where the interaction is needed,” Fanning said. “The question then becomes 'how does it work?'”
For pol-prim to start laying down RNA primers, hRPA must detach from the ssDNA. With the new information about the binding between Tag and hRPA, Chazin and Fanning developed a model that may explain this early mechanism of DNA replication.
“We had previously shown that binding of Tag to RPA can scrunch it up, creating some ssDNA transiently,” Fanning said. In this brief moment when ssDNA is exposed, Tag guides the pol-prim enzyme, which is bound to Tag in a different location than hRPA, into the space vacated by hRPA, and DNA replication can commence.
Not only does their model explain the very early molecular events in DNA replication, but it may help researchers pin down the mutational defects that lead to cancer and suggest possible targets for anticancer therapies.
“Behind tumor formation is the process of cell transformation — where a cell acquires the unlimited capacity to replicate and grow,” Chazin said. “There's a direct link between SV40 and cell transformation. We would like to work out some of this basic biology in order to understand the complexity of tumor formation because, if we can get to the bottom, we can attack cancer at a much more fundamental level than the way it's being addressed now.”
And to understand a complex process like cancer, collaborations like the one Chazin and Fanning have developed will become ever more important.
“This is a great example of the kind of interdisciplinary research that NIH is calling for — truly integrated research where we exchange ideas on a regular and integrated basis,” Chazin said.
“The success that Ellen and I have had exemplifies one of the mandates that we have in setting up the Center for Structural Biology — to spread the power of structural thinking and the potential of structural biology projects as widely as we possibly can throughout the communities of biology and medicine.”
“We've been through all kinds of ups and downs in this project and there's no doubt more to come,” Fanning said.
Other Vanderbilt authors on the paper include Alphonse Arunkumar, Vitaly Klimovich, Xiaohua Jiang, Robert Ott, and L. Mizoue. The research was supported by the National Institutes of Health, Vanderbilt-Ingram Cancer Center, Vanderbilt Center in Molecular Toxicology, the Howard Hughes Medical Institute Professors Program, and Vanderbilt University.
Fore more information, visit the HHMI Web site: http://www.hhmi.org/news/ fanning.html.