University of Georgia Scientists Discover Gene that maintains Genome Integrity by Limiting DNA Repli

ATHENS, Ga. – A team of cell biologists at the University of Georgia, led by Edward Kipreos, has discovered a gene that maintains genome stability by controlling the level of DNA replication.

The discovery, while still at the level of basic science, could have important implications in future studies of the genome instability present in cancer cells, and in particular, the gene- amplification “events” that often trigger cancer.

“The replication of DNA is strictly regulated to occur only once per cell cycle,” said Kipreos. “We found that the loss of a gene named CUL-4 completely abolishes this regulation. In cells lacking CUL-4, the replication of DNA is continuously re-initiated during the same cell cycle to produce cells with greatly expanded levels of genomic DNA.”

The research was published today in the journal Nature and was supported by a grant from the National Institutes of Health. Co-authors of the paper are graduate students Weiwei Zhong and Hui Feng, and technician Fernando Santiago, all of the Kipreos lab.

Using the tiny worm called Caenorhabditis elegans (the common nematode found in soils all over the world) as a model organism, the team found that CUL-4 controls proper DNA replication by promoting the degradation of a protein called CDT-1 that is required for the initiation of DNA replication.

There are five genes in the cullin family, CUL-1 through CUL-5, and they are involved in the degradation of other cellular proteins. They have been known for less than a decade, but scientists do know that the over-expression of the CUL-4 gene in humans plays a role in both breast and liver cancers, so knowledge of how CUL-4 controls DNA replication could open new areas of investigation for cancer researchers.

The current study expands on research from other scientists, including work from the Imperial Cancer Research Fund published in September 2001 in The EMBO Journal. Using yeast as a model organism, a team led by Nobel laureate Paul Nurse found that the limited expression of two “DNA replication licensing proteins,” CDT-1 and CDC-18, is crucial to ensure a single cycle of DNA replication.

Work by the Kipreos lab published today in Nature shows that CUL-4 controls the CDT-1 replication licensing protein by removing it from cells that are duplicating their DNA. When CDT-1 is not degraded in replicating cells, its presence causes the re-initiation of DNA replication.

The University of Georgia team used a technique called RNA-mediated interference (RNAi) to probe the function of CUL-4. The basis of the technique involves injecting double-stranded RNA (dsRNA) into an organism, thereby inactivating the gene corresponding to the dsRNA. In this way, the researchers inactivated CUL-4 and were able to study cell division in its absence.

What they discovered was a dramatic increase in the size of “blast” cells, which are cells that proliferate in the developing C. elegans larvae.

“We measured the amount of genomic DNA in the enlarged blast cells and we were amazed at the extent of the genomic DNA expansion, which can be up to 50 times the level of normal cells,” said Kipreos.

The researchers knew that three mechanisms could be generating the dramatic increase in genomic DNA levels. These mechanisms include failed mitosis; endoreplication, in which cells bypass cell division and enter the next cell cycle with doubled DNA; and re-replication, in which cells remain “stuck” in one cell cycle phase and continuously re-initiate DNA replication.

Using molecular techniques, Kipreos and his colleagues found that DNA was amplifying by re-replication in the cells lacking CUL-4, resulting in markedly increased genome sizes. CUL-4 is the first known example in which the loss of a single gene produces such completely unrestrained DNA replication.

This suggests that CUL-4 acts as a guardian of the genome to protect it from unrestrained replication. A failure to limit DNA replication leads to the indiscriminate expansion of genomic DNA, which has catastrophic consequences for the survival of the organism.

The identification of CUL-4 as a major regulator of DNA replication levels will likely lead to investigations in mouse and human models and could open new studies for how DNA replication goes awry in numerous disease processes.