DNA Fidelity And Genomic Instability

NIEHS - Div. of Extramural Research and Training Annual Report 1997

In eukaryotic cells, DNA processing (e.g., transcription, replication, and repair) occurs in DNA packaged in chromatin. The fundamental unit of chromatin structure, the nucleosome, is involved in specific gene repression of gene activation. It is believed that nucleosomes are able to repress genes by blocking the access of trans-acting factors to their binding sites in gene promoter regions. Agents that alter the binding (or positioning) of these nucleosomes could affect the ability of nucleosomes to repress or facilitate gene expression.

For several years, Dr. Michael Smerdon, from Washington State University, has investigated the effects of DNA damage by chemicals and UV light on the formation of a positional nucleosome. His results showed that a single adduct of a model xenobiotic can enhance the level of nucleosome damage in vivo, where this damage is spread throughout the genome with frequency of about one adduct per 100,000 base pairs. Such a change in nucleosome stability could have significant consequences if damage occurs in a critical location (e.g., gene promoter). Within a transcribed region of a gene, a more stable nucleosome could affect nucleosome disruption during transcription, or transcription-coupled repair, possibly leading to truncated RNAs or mutation, and ultimately to genomic instability.

Gene rearrangements and genomic instability are often a consequence of exposure to many types of environmentally-induced damage. Of particular relevance to environmental health is the increasing exposure of large populations to ultraviolet (UV) light. UV-induced DNA damage and the mutagenic and carcinogenic effects of short-wave UV light have been well established in all organisms that have been investigated, including humans. Furthermore, there is a well established link between inefficient DNA repair of UV damage and carcinogenesis in humans, as demonstrated through the study of the human disease, xeroderma pigmentosum. Also, there exists a significant literature relating the mutagenic and carcinogenic effects of sunlight exposure to the formation and persistence of various UV light- induced dipyrimidine DNA photoproducts. Although DNA repair enzymes have been discovered that monitor DNA for such damaged sites and subsequently initiate repair, the active sites and chemical reaction mechanisms for these enzymes have previously remained unknown.

During the past year, Dr. Stephen Lloyd, from the University of Texas Medical Branch at Galveston, has made considerable progress in understanding the function of enzymes that initiate the removal of ultraviolet light-induced, mutagenic and carcinogenic DNA lesions. One of the major unsolved problems in this area is how abnormal DNA structures are recognized relative to undamaged DNA. In regard to this problem, they examined the initial steps in the base excision DNA repair pathway for evidence that enzymes slide along DNA and "flip" bases out of their normal position within the DNA duplex in order to determine if they are damaged. In this way, enzymes are capable of "sampling" DNA for its relative integrity. As a damaged base is being flipped to an extrahelical position, it may bind into an exclusive pocket in the cleft on the enzyme. Alternatively, some enzymes flip bases that are opposite to the damage base, and as a result, they insert their catalytic active site in the hole in the DNA that is created in close proximity to the damaged base. Using fluorescent DNA probes to follow this mechanism for several DNA repair enzymes, the researchers have found that the process of base flipping appears to be a common feature of these proteins. Thus, this laboratory has developed not only a unified catalytic mechanism for DNA glycosylases, but also a unified mechanism for the steps preceding catalysis (Lloyd, R01ES04091).

Understanding repair of DNA in specific regions of the packaged structure in the cell nucleus is crucial to understanding why certain DNA lesions are not repaired for long times in human cells. Such "long-lived" lesions can form mutations and ultimately lead to cancer and other debilitating diseases. Furthermore, selective repair of certain chromatin domains has been shown to be absent in some of the repair deficient human diseases associated with increased cancer frequency (Smerdon, R01ES02614).

For example, the human syndrome of hereditary nonpolyposis colorectal cancer, HNPCC, has been linked to germline mutations in the human homologs of E. coli and yeast DNA mismatch repair (MMR) genes. This syndrome is associated with a high risk of early-onset colon cancer, as well as cancers of the endometrium and other sites. In addition, evidence is emerging that the MMR gene homologs may participate in various other cellular functions, such as transcription-coupled repair, recombination, and even cell cycle regulation. Consequently, a deficiency in one of these homologs may disrupt genome stability in multiple ways.

In an attempt at gaining a better understanding of how the DNA repair mechanism works; Dr. Peter Glazer, from Yale University, has been working for the past five years in the generation of a novel colony of transgenic mice. He has created transgenic mice carrying several different mutation reporter genes, which have proved to be quite useful for studying mutagenesis and genomic instability in a whole animal model. Even more importantly, he has constructed a series of hybrid mouse lines that not only carry the reporter genes but also carry targeted disruptions of selected DNA mismatch repair genes. His research team has made knock-out mice deficient in the PMS2, MLH1, and MSH2 MMR genes. Transgenic mice with targeted disruptions of selected MMR genes are cancer prone and have spontaneous mutation frequencies that are up to 100 fold elevated above that of the wild type. Consequently, these animals are of particular interest to the scientific community for the study of carcinogenesis and mutagenesis (Glazer, R01ES05775).

Evidence is accumulating that the activities of the MMR factors are not limited to mismatch repair. Mice lacking PMS2 or MLH1 function, show specific patterns of infertility, suggesting a role for these factors in meiosis. Researchers have demonstrated that human cancer cells deficient in MMR exhibit slower than normal removal of UV damage from the transcribed strand of active genes, implying that efficient transcription- coupled repair in some manner requires the MMR system. It was also found that MMR-deficient cells exhibit mildly decreased survival following UV irradiation. In contrast, cells deficient in MMR demonstrate significantly increased survival relative to wild type upon exposure to alkylating agents. This alkylation tolerance in cells lacking MMR is thought to reflect a role for MMR in mediating the toxicity of alkylating damage. By one model, the MMR pathway may recognize mispairs formed during replication of an alkylated template. The system initiates futile attempts to repair the undamaged strand, leading to excessive strand breaks and consequent lethality. Cells without active MMR do not attempt this abortive repair and are spared the full toxic effects of alkylation damage. Therefore, the MMR system in mammalian cells can influence the cellular response to a variety of lesions other than mismatches and appears to participate in multiple repair pathways.

DNA mismatches, including base mispairs and loop-outs of various sizes, can come about by several mechanisms, including replication errors, repair errors, or as intermediate in recombinational processes. Repair of such errors is critical to the maintenance of genomic integrity.

DNA repair is a ubiquitous phenomenon that evolved to counter pathological consequences of abnormal DNA structures due to the presence of damaged, unusual and mispaired bases. In fact, DNA repair may be essential for organism survival, and is a key element in the intricate web of pathways the cell uses to prevent genomic instability.

source: http://www.niehs.nih.gov/dert/annrpt.htm 

Mindfully.org note: notations such as  (Glazer, R01ES05775)  are the names of the recipients and their NIEHS research grant numbers.