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PATHOLOGY Molecular Bureaucrat Tied to Replication Approval Process Before DNA replication can occur in a eukaryotic cell, the nucleus must be licensed for replication by a complex bureaucracy of proteins. Once replication begins, these proteins quickly shut the door on further licensing because duplicate rounds of replication could lead to genomic instability and cancer. Cdt1 helps form the pre-replication complex that attaches to DNA during the G1 phase of the cell cycle. Geminin and Cdt1 are expressed in different periods of the cycle. But when both are expressed in the S phase (shaded in pink), geminin prevents re-replication by inhibiting Cdt1. Adapated from original by Anindya Dutta Method Breaks Down, Builds Up DNA Replication A cherished notion in cell biology has held that the nucleus functions as something of a magic box, its overarching structure providing a necessary scaffold for eukaryotic DNA replication to occur. But Johannes Walter overturned that assumption, not so much by thinking outside the box as by throwing it away completely. Walter, an HMS assistant professor of biological chemistry and molecular pharmacology, developed a cell-free system for DNA replication without a nucleus, using a concoction of Xenopus egg extracts and a dose of highly concentrated nuclear components. This recipe for replication has been surprisingly effective, suggesting that it is not the nucleus itself but the ingredients within it that allow replication. The system can now be used to study the proteins involved in a systematic way. "Because we don't have a nucleus, all manipulations become easier," he said. An in vitro system is a crucial part of Walter's larger goal: to take apart the process of DNA replication piece by piece until every bit is defined—and then put it back together again. "The only way to rigorously show that you have everything in hand that is necessary to carry out a biochemical reaction is to reconstitute it from purified components," he said. Studies have identified numerous factors involved in initiating replication, but there has been no way to know if all the factors have been identified, or exactly how they interact and in which step of the process. Sound advice for approaching a daunting task is to break it down into reachable goals and discrete steps. In the Dec. 15 Journal of Biological Chemistry, Walter shows that two major protein kinases that help initiate replication act in a specific sequence rather than together. This finding makes his work a little easier by providing two discrete steps that can be reconstituted separately, rather than one complex step. Walter's lab is now working on reconstituting the second of these steps, catalyzed by the protein kinase cdk2/cyclin E. He is confident that by systematically identifying each component of the extract needed to carry out this reaction, his approach will finally yield the substrates of cdk2/cyclin E, one of the coveted goals of cell cycle research. Standard Procedure A team led by Anindya Dutta, HMS associate professor of pathology at Brigham and Women's Hospital, and James Wohlschlegel, a BBS student in his lab, has uncovered one of the mechanisms cells use to shut down the licensing process after replication starts. In the Dec. 22 Science, they report that the replication inhibitor geminin targets Cdt1, one of the factors necessary for replication. This inhibitory pathway seems to work in parallel with other regulatory processes to prevent re-replication and keep the cell cycle running smoothly. The protein machinery required to initiate DNA replication assembles at specific launch sites, or origins, along the DNA helix. An initial group of proteins called the origin recognition complex binds to origin sites and recruits regulatory molecules CDC6 and Cdt1 which, in turn, recruit another set of proteins, the mini-chromosome maintenance (MCM) complex. All of these molecules form the pre-replication complex, a kind of launch pad for DNA replication. Once the machinery fires, this launch pad must be quickly disassembled to prevent further launches that would cause sections of DNA to be copied more than once. Discovered in 1998 in the lab of Marc Kirschner, the Carl Walter professor and chair of cell biology at HMS, geminin was believed to inhibit replication by preventing the MCM complex from binding to replication origins, but how it related to other proteins in the licensing process was unknown. Dutta credits Wohlschlegel for drawing the logical connection between geminin and the newly discovered protein Cdt1, which seems to be a necessary factor for recruiting the MCM complex in Xenopus DNA replication. "Addition of geminin blocked replication at the same stage as depletion of Cdt1. James was the first one who connected one and two and said, 'Aha, geminin might be targeting Cdt1.'" Anindya Dutta (left) and James Wohlschlegel (center) collaborated with Johannes Walter to figure out how geminin helps guard against re-replication of DNA. Photo by Jeff Cleary The team identified the human Cdt1 protein and determined that geminin interacted with Cdt1 and vice versa. But to confirm the relationship between Cdt1 and geminin, they turned to Johannes Walter's lab, which had developed an in vitro system for studying the factors involved in DNA replication (see sidebar). "All we had shown was that they interacted," said Wohlschlegel. "We needed a better way to show that geminin interacting with Cdt1 actually had some sort of biological meaning." Using Walter's in vitro assay with Xenopus egg extracts, the team showed that the inhibition of replication by geminin was blocked by adding Cdt1 and that Cdt1 was the likely target of geminin. Splitting Up The cell cycle is a highly orchestrated drama in four acts. It begins with the G1 phase, the normal resting phase of a cell, crescendos into the S phase, when DNA replication occurs, pauses in the G2 phase to collect its chromosomes, and then culminates in the M phase, when mitosis divides the cell in two. Directing the entire assemblage of proteins are cyclin-dependent kinases that wax and wane according to the cell cycle. These kinases have the dual role of initiating replication and, as their levels rise after replication initiation, blocking re-replication by preventing the launch pad of proteins from reforming. All of the proteins that play a role in this process are strictly regulated, with specific entrances and exits. For instance, geminin was thought to be involved in inhibiting replication because it appears after replication begins in the S phase but is degraded after mitosis. Cdt1 has the opposite role, appearing in the G1 phase when replication factors are assembling but disappearing in the S phase. The fact that these two separately regulated factors also directly interact with each other suggests that eukaryotic cells have developed an additional mechanism to prevent re-replication. If something happened to disrupt the activity of cyclin-dependent kinases, such as damage to the DNA, the direct inhibition of Cdt1 by geminin would still keep the show running by preventing re-replication despite the absence of the director. Kirschner says that the paper, by tying geminin directly to a replication protein, confirms the suspicion that geminin plays an inhibitory role in licensing a cell for replication. —Courtney Humphries