Environmental Factor, June 2008, National Institute of Environmental Health Sciences
MIT Researcher Offers Insight into Death by DNA Repair
By Robin Arnette
On May 19, the Genetics Training Action Committee (TAC), comprised of postdoctoral fellows in the Laboratory of Molecular Genetics, hosted a talk by DNA damage expert Leona Samson, Ph.D. The presentation, "Complex Responses to Damaging Agents," was part of TAC's continuing seminar series and took place in Rodbell Auditorium. IRTA Fellow Chris Halweg, Ph.D., hosted the event.
As director of the Center for Environmental Health Sciences and professor in the Department of Biological Engineering at the Massachusetts Institute of Technology, Samson(http://web.mit.edu/be/people/samson.htm) studies the health effects of alkylating agents, chemical compounds containing reactive moieties made of carbon and hydrogen atoms called alkyl groups. Because alkylating agents can easily combine with other molecules, they induce different kinds of damage to DNA, but these attacks and how the damage is dealt with are modified by the organism's genetic susceptibility. Furthermore, it turns out that the act of repairing damaged DNA can sometimes lead to mutations and cell death, instead of preventing mutation and death. Samson devoted the majority of her talk to two examples of what she calls death by DNA repair at N3-methyladenine (3MeA) and O6-methylguanine (O6MeG) lesions. The studies took place in mouse models and are unpublished.
In the case of an alkyl-induced 3MeA lesion, which blocks replication, base excision repair (BER) is employed and mammalian 3MeA DNA glycosylases remove the lesion. Sampson wanted to know what would happen if the glycosylases weren't present. "Bevin Engelward, when she was student in the lab, made a double knockout of mouse embryonic stem cells, removing the 3MeA DNA glycosylase, and we got what we expected: the absence of repair of replication blocking lesions," she said. They went on to make the knockout mouse, "but surprisingly, the bone marrow cells from these Aag null glycosylase-deficient mice were resistant to being killed by the alkylating agent methyl methanesulfonate (MMS)."
Samson offered an explanation for the resistance by saying that wild-type glycosylase levels lead to the accumulation of toxic BER intermediates that can stimulate cell death. In the absence of that glycosylase, one doesn't produce these BER intermediates and the cells can become resistant. Other studies with glycosylase-deficient animals confirmed that retina and cerebellum cells were also resistant to alkylation.
In regard to O6MeG lesions caused by alkylating agents, Samson's team had to examine a different mechanism. The O6MeG lesion doesn't block DNA replication like 3MeA does; it tricks the DNA polymerase into inserting a thymine opposite of the modified guanine. A protein called methyltransferase transfers the methyl group from the O6-guanine onto a cysteine residue 15 in the active site, thereby restoring a normal guanine in DNA. This action prevents mutations induced by the O6 lesion.
Samson found that if she introduced the methyltransferase gene into human cells, the cells became resistant to alkylating agents. "This was confusing because O6MeG is a mispairing lesion," Samson explained. "Why would it toxic to the cell?" It took a few years, but work with many alkylation-resistant cell lines provided the answer she was looking for.
It turned out that the cells didn't become resistant by acquiring methyltransferase repair activity; they'd become resistant because they had lost activity in the mismatch repair pathway. The mismatch repair pathway mediates the toxicity of O6MeG lesions. Using knockout mice, Samson's group provide in vivo evidence that the Exo1 nuclease (known to take part in mammalian mismatch repair) is required for most of the apoptosis induced by O6MeG lesions.
Samson said the ultimate outcome of being exposed to an alkylating agent or any environmental agent ends up being a balance of many different factors, even among different repair pathways.