Researchers at Cold Spring Harbor Laboratory have discovered the protein that controls the copying of the centrosome in human cells and prevents it from being re-duplicated. The molecule, called ORC1, is among the six proteins that comprise the origin recognition complex (ORC), an assembly that attaches to particular sequences within all DNA in the cell and prepares it for duplication.
The scientists revealed that Orc1 additionally controls the number of centrioles in a cell. It seems to accomplish this feat by keeping the pair of centrioles in each centrosome connected to one another. Upon the cell's commitment to cell division, however, the centriole pair is duplicated to produce two new centriole pairs, one in each centrosome; this occurs precisely as copying of the chromosomes gets under way.
Cells that lacked Orc1 exhibited centrioles that were disengaged from the original, suggesting that Orc1 might prevent re-duplication by helping the new centrosomes to stay connected to the old.
To determine which of the ORC proteins limits centrosome copying, the team used RNAi to block the production of each of the proteins that combine to form ORC in human cells. Loss of Orc1 alone, the scientists found, spurred cells to accumulate excess centrosomes.
Cells that were induced to produce more Orc1 had the normal amount of centrosomes even when centrosomes were forced to re-duplicate via drug treatment of cells. The scientists thus deduced that Orc1 must allow cells to duplicate centrosomes once per division cycle but prevents centrosomes from being re-duplicated.
These new roles for Orc1 are separate from its duty to help jump-start DNA duplication. The CSHL team found that a shortened version of Orc1 that lacked the ability to start DNA duplication was still able to limit centrosome copying to once per cell-division cycle.
Regarding the ability of Orc1 to control the number of centrioles, the researchers showed that it depend on the protein’s ability to physically associate with the centrosomes. Though investigators believe Cyclin A moves Orc1 to the centrosomes, Cyclin E may be the target of Orc1. They found that Orc1 antagonizes Cyclin E activity when it is still present in the cell so that it duplicates centrosomes but cannot re-duplicate them.
The scientists thus propose that Orc1 enforces the number of centrosome copies by moving to centrosomes during the short temporal window in the cell division cycle when Cyclin E is still present in the cell. “During this time, if the effects of Cyclin E activity aren't counteracted by Orc1, centrosome re-duplication can occur,” explains Bruce Stillman, Ph.D., professor at CSHL.
The findings appear in the February 6 issue of Science.
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