The process of DNA replication has stringent controls to ensure that the genome does not go awry from significant errors. However, what results from the loss of these controls has not been examined until very recently.
Researchers collaborating from the Hopkins School of Medicine and University of California, San Francisco, have tested the impacts of interference with eukaryotic DNA replication controls. By manipulating the genome of yeast cells to create additional copies of particular segments of DNA, the group was able to increase the expression of particular segments. The group notes that this may have broader implications for gene copy number changes in the research of cancer biology, human genetics and evolution.
The budding yeast Saccharomyces cerevisiae, commonly used in the fermentation of alcoholic beverages and baking, was manipulated by the researchers so that the controls that prevent the re-replication of a particular genetic sequence were disrupted. By deregulating two important proteins involved in the regulation of DNA replication in one of their trials, the researchers managed to undermine these controls in such a way as to induce re-replication.
Interfering with these controls during a particular window of the cell cycle resulted in re-replication of a specific segment of DNA that was not appropriate for the yeast, causing multiple copies of the segment to develop. The production of these additional copies resulted in a significantly higher frequency of expression of these segments.
These changes were observed using PCR and Southern blot analysis, indicating which segments were amplified and to what degree. Certain instances of induced re-replication resulted in increases in expression by as much as 48-fold.
The researchers also noted a possible mechanism for re-replication where the extra replication strand is forked with the other copy of the segment, resulting in possible damage to the segments. Both strands break and reconnect end to end resulting in an elongation of the gene segments with possible defects in the re-replicated strands. Specifically, these segments are arranged in a head-to-tail pattern.
From these results, the group presents another important genetic event that will be of great importance for human geneticists, evolution biologists, and cancer researchers.
“Our work in budding yeast raises the possibility that loss of replication control in more complex organisms might contribute to a number of normal and pathological processes where gene amplification is observed,” wrote Joachim Li, associate professor in the Department of Microbiology and Immunology at UCSF, in an email to the News-Letter.
Li also noted that there is a great significance of this re-replication for cancer research, especially in regard to anti-tumor treatments. “Amplification of oncogenes is thought to contribute to oncogenesis, and amplification of specific genes can lead to resistance to anti-tumor drugs,” he wrote.
With regards to evolution, gene duplication may have a large impact when intertwined with the role of mutation. “Increasing the copy number of genes in itself may provide phenotypic variability upon which natural selection can work and where simple gene duplication can allow genes to acquire new functions by mutation,” he wrote.
The group also noted that the high efficiency of re-replication resulting from the loss of DNA replication control in its ability to destabilize a genome, noting that even at lower levels of re-replication there is a significant amount of resultant gene amplification.
The loss of DNA replication control may have an impact on further studies in cancer and evolution research. “Demonstrating that loss of replication control can indeed affect cancer or evolution is one of our new directions,” Li wrote.
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