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Yeast Model Used to Explore Genomic Hypermutation

By Brian Chorley
January 2009

Visiting Fellow Yong Yang
Visiting Fellow Yong Yang was first author on the study. (Photo courtesy of Steve McCaw)

Gordenin, above, also worked with Chromosome Stability Group
Gordenin, above, also worked with Chromosome Stability Group co-authors Biologist Joan Sterling and former Postdoctoral Fellow Francesca Storici, Ph.D., who is now with the Georgia Institute of Technology. (Photo courtesy of Steve McCaw)

Chromosome Stability Group Head Mike Resnick
Chromosome Stability Group Head Mike Resnick is NIEHS Scientist of the Year. (Photo courtesy of Steve McCaw)

A recent discovery by researchers at NIEHS sheds new light on the mechanisms of the unintentional changes in DNA that may lead to cancer and genetic disease. The Chromosome Stability Group, led by Michael Resnick, Ph.D., in the Laboratory of Molecular Genetics, found common sources of DNA mutation that persist with high tolerance and may predispose individuals to genetic disease. The group's study ( Exit NIEHS, featuring Visiting Fellow Yong Yang, Ph.D., as first author and Staff Scientist Dmitry Gordenin, Ph.D., as principal investigator, appeared in the open-access journal PLoS Genetics.

Unintended genomic mutations can arise from multiple sources, including errors in DNA replication and repair and environmental DNA damage - usually with no obvious consequences. Some mutations, however, may be deleterious or beneficial to an organism by increasing or decreasing adaptation to the environment and may be passed to offspring. Mutation, therefore, performs an important role in evolution.

Some mutations are important to normal biological function. For example, the immune system utilizes a highly regulated process strictly confined to immunoglobulin locus known as somatic hypermutability, which generates variable recognition sites in a subpopulation of B-cells that recognize foreign antigen with different affinities. B-cells with receptors for best affinity to a particular antigen are then selected for an individual's immune response. Importantly, the rest of the genome is free from a mutation burden.

Other sites of unregulated localized hypermutability are known to exist. Regions of the genome that have undergone double-strand break repair exhibit higher frequencies of mutation. Additionally, dysfunctional chromosomal ends, known as "uncapped" telomeres, indicated hypermutability in yeast. Common intermediates in these events are long stretches of single-stranded DNA (ssDNA). Since prevention of DNA mutation depends on repair mechanisms that act primarily on double-stranded DNA, it has been theorized that ssDNA is a likely target of hypermutation.

To address this theory, the research team developed two sophisticated yeast models that allowed selection of subpopulations that had undergone double-strand break repair or transient uncapping of a telomere. These subpopulations were exposed to UV light or a chemical mutagen, methyl methanesulfonate, which induced DNA damage and subsequent mutation. Types and frequency of mutations were then assessed by reporter genes incorporated into the model yeast strains.

The study found that multiple types of mutation occurred in ssDNA with frequencies reaching one per 400 to 800 bases of DNA. This high mutation frequency is comparable to somatic hypermutability in immunoglobulin genes. In addition, the researchers found that mechanisms which direct hypermutability in immunoglobulin genes also direct hypermutability in ssDNA. They then used yeast strains deficient in specific components of DNA repair to verify that the error-prone polymerase ΞΆ activity was essential.

Importantly, yeast survival was high in populations that exhibited hypermutability. The incidence of genome-wide mutation was low compared to regions near the double-strand break after mutagen exposure. Cellular mechanisms in place that prevent accumulation of wide-spread genomic mutation did not recognize the localized mutation in these colonies.

Circumstances where ssDNA occur in yeast also exist in mammalian cells. This is true both for double-strand breaks and during periods of DNA replication and gene transcription. Yang speculated that hypermutability of ssDNA is a mechanism for genomic mutation in humans and suggested this process has consequences for predisposition to genetic disease - notably cancer. Model studies such as these may therefore lead to association of human disease with hypermutation susceptibility. "Human health is our final goal," stated Yang.

The researchers hope to develop their tool for use in a semi-high-throughput manner. Yang explained that this would allow more rapid identification of chemical mutagens amenable to hypermutation. Gordenin added that in addition to meticulously built model systems, the development of highly efficient technologies for whole-genome sequences opens new opportunities for investigators. This development will allow researchers to trace multiple mutation tracks left by transient localized hypermutability all over genomes and to evaluate environmental damage to genes and detect alterations that may lead to disease susceptibility.

Citation: Yang Y, Sterling J, Storici F, Resnick MA, Gordenin DA ( Exit NIEHS. 2008. Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae. PLoS Genet 4(11):e1000264. Epub 2008 Nov 21.

(Brian Chorley, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Environmental Genomics Group.)

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