Oct. 5, 2001 LOS ALAMOS, N.M., Oct. 4 2001 - Biologists at the U.S. Department of Energy's Los Alamos National Laboratory have discovered new insights into how two common proteins found in mammalian cells can cause chromosomes to fuse together -mutations that can destroy cells or give rise to cancer.
The research, by Susan Bailey and Edwin Goodwin of Los Alamos' Biosciences Division, was published recently in the journal Science.
Bailey, Goodwin and their colleagues looked at the role of telomeres in protecting chromosome ends. Chromosomes are made of deoxyribonucleic acid - DNA - and are the carriers of genetic information. Human cells contain 22 pairs of chromosomes plus two gender chromosomes. Telomeres are specialized protective structures at the end of each arm of the X-shaped chromosomes.
Without telomeres, natural chromosome ends appear to the cell like broken DNA ends in need of repair. Mutations in certain genes impair the protective function of telomeres leading to inappropriate "repair," in effect causing chromosomes to fuse together end to end. Chromosome end fusions destabilize the orderly transmission of genetic information to the next generation of cells. The Los Alamos researchers studied telomere dysfunction in order to learn more about how a normal telomere works.
Each chromosome has four telomeres. Mammalian telomeres contain a unique DNA sequence, discovered earlier by Los Alamos' Human Genome Project, as well as specialized proteins that together create a protective cap at the ends of chromosome arms.
Bailey and Goodwin looked at the role of two proteins in telomere function. One protein was known to play a role in telomere function and chromosome end capping. The other protein originally was shown to help repair damaged DNA, but later was shown by Bailey and Goodwin to also help protect natural chromosome ends.
The researchers used human cells provided by Titia DeLange of Rockefeller University that contained artificially induced changes to the first protein and mouse cells with mutations in the second protein. Under normal circumstances, when cells divide they produce exact duplicates, including exact duplicates of the chromosomes they contain. Bailey, Goodwin and their colleagues found that the progeny of cells containing the altered proteins often contained chromosomes that had fused with other chromosomes at one arm. The fused chromosomes had a sausage-like appearance and were easy to distinguish from normal chromosomes. Due to their genetic abnormalities, the damaged daughter cells often were unable to thrive.
The fusing chromosome arms in the dying daughter cells indicated that the malfunction might be associated with telomere replication and indicative that the protein changes induced in the original cells played a role. But Bailey and Goodwin noticed something else - something extraordinary and unexpected.
Using a Los Alamos-developed technique called chromosome-orientation fluorescence in situ hybridization - CO-FISH -that highlights which half strand of the DNA double helix underwent replication during the cell-division process, the researchers determined that the fusion only occurred on specific arms of the chromosomes. What's more, Bailey and Goodwin noticed that fusion never occurred in a chromosome on two arms on the same-side of the "X"; if more than one fusion occurred in a single chromosome, the fusion always occurred on opposite arms on opposite sides. This indicated to the researchers that not all telomeres in a chromosome are the same, because if they were, the researchers would have expected to see same-side fusion in at least some cases simply based on the laws of chance.
"A lot of research has been done on telomeres in the biological community, and the conventional thought was that all telomeres are created alike," said Goodwin. "Our research shows that this is not the case. There are two different processes for protecting telomeres and they have distinct genetic requirements."
The difference in telomeres apparently lies in the way chromosomes are replicated, Bailey and Goodwin found. When chromosomes duplicate themselves, their DNA double helices separate into two strands and then rebuild their DNA structure on each half strand. Because DNA polymerases - the protein catalysts that make the new DNA strands - proceed in only one direction, the two new telomeres replicated from the original parent telomere are produced by two different mechanisms.
In one case, the telomere's double helix terminates in a blunt end. In the other case, the telomere ends with a minute chemical overhang. This overhang is important because it allows the telomere to loop back on itself - forming a so-called "t-loop" - to complete its end cap. The first process is known as leading-strand DNA synthesis; the second is known as lagging-strand DNA synthesis.
Bailey, Goodwin and their colleagues found that chromosome fusion occurred only at sites on the chromosomes where telomeres had been formed by the leading-strand process.
The research indicates that both altered proteins induced in the cells used for study play a role in capping the ends of telomeres formed by leading-strand DNA synthesis, but are not required to cap telomeres replicated by lagging-strand synthesis.
Bailey and Goodwin's research is significant because it has shown the existence of two types of telomeres and also gives insight into the roles of two proteins in normal cell function.
All of Bailey's and Goodwin's co-authors of the paper are former Los Alamos researchers, and include: Michael Cornforth, Department of Radiation Oncology, University of Texas Medical Branch; and Akihiro Kurimasa and David Chen, Cell and Molecular Biology Division, Lawrence Berkeley National Laboratory.
Los Alamos National Laboratory is operated by the University of California for the U.S. Department of Energy's National Nuclear Security Administration.
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