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Translesion DNA Synthesis Uses Specialized Polymerases

By Robin arnette
January 2008

Friedberg began his talk by describing DNA damage as the
Friedberg began his talk by describing DNA damage as the "scourge of all living organisms." (Photo courtesy of Steve McCaw)
Co-host Pursell introduced the lecturer, taking care to pronounce the tongue twister in the speaker's alma mater, the University of the Witwatersrand. Following the question-and-answer session, Pursell showed his enthusiasm over Friedberg's talk.
Co-host Pursell introduced the lecturer, taking care to pronounce the tongue twister in the speaker's alma mater, the University of the Witwatersrand. Following the question-and-answer session, Pursell showed his enthusiasm over Friedberg's talk. (Photo courtesy of Steve McCaw)
Pursell's mentor and lecture co-host Kunkel help field questions from the audience.
Kunkel, Pursell's mentor and lecture co-host, helps field questions from the audience. (Photo courtesy of Steve McCaw)

On December 11, Errol C. Friedberg, M.D., delivered a seminar about DNA damage and repair to a near capacity audience in Rodbell Auditorium. The seminar, titled "Specialized DNA Polymerases in Higher Organisms: Insights from the Pol? [polymerase kappa] Knock-Out Mouse," was part of the NIEHS Distinguished Lecture Series. Principal investigator Tom Kunkel, Ph.D., and Fellow Zachary Pursell, Ph.D., of the Laboratory of Molecular Genetics hosted the event.

Friedberg (http://webpath.swmed.edu/research/FMPro?-db=labname.fp5&-format=rlab%5fmain.html&-lay=cgi&-sortfield=labname%5ft&error=error.html&divisionID%5fc=1&-recid=8&-token.0=1&-find) Exit NIEHS, holder of the Senator Betty and Dr. Andy Andujar Distinguished Chair in Pathology, is also professor and chairman of the Department of Pathology at the University of Texas Southwestern Medical Center in Dallas, Texas. His research focuses on understanding the molecular mechanism of DNA repair and mutagenesis in eukaryotes and the roles these processes play in cancer.

Friedberg prefaced his talk by providing background on how organisms handle molecular damage that halts DNA replication. "Through eons of evolution, cells have relied upon a variety of DNA damage tolerance mechanisms," he said. "Lesions that were not repaired or removed from the genome were, in fact, tolerated so that important phenomena like replication and transcription could continue past a bulky DNA template lesion." Friedberg emphasized that repair would occur somewhere down the line, but how these tolerance mechanisms worked remained a mystery until about 10 years ago.

That was when a new class of enzymes-Friedberg prefers to call them specialized polymerases-was discovered. A specialized polymerase differs from a high fidelity polymerase in that a specialized polymerase allows an open and solvent-accessible active site that can tolerate lots of lesions. In contrast the tight structure of a high fidelity polymerase permits the DNA duplex to fit snugly into its active site.

Specialized polymerases are involved in translesion DNA synthesis (TLS), one of the tolerance mechanisms Friedberg is interested in, and research from several labs has demonstrated that multiple polymerases, such as Rev1 protein, polymerase iota (Pol?) and polymerase eta (Pol?), work together during this process. "When the replication machinery is arrested, a polymerase switch is believed to occur," Friedberg explained. "Rev1 will incorporate one or, at most, two cytosine residues, irrespective of what the nucleotide structure of the template strand is, but if a thymine dimer is present, Pol? incorporates a small number of nucleotides which allows bypass of the arresting lesion."

Friedberg's work may suggest that specific specialized polymerases evolved to bypass particular lesions during TLS. He and colleagues determined that Pol?, a newly discovered specialized polymerase, bypasses bulky polycyclic or multiple-ring adducts on DNA. The experiments compared cells from normal mice and cells from a strain of knock-out mice that lacked Pol? (pol?-/-). Benzpyrene-a five-ring aromatic hydrocarbon mutagen found in coal tar, cigarette smoke, jet fuel exhaust and charcoal-broiled meats-served as the lesion.

In collaboration with a group from the Weitzman Institute in Israel, Friedberg and his colleagues generated a plasmid that had a benzpyrene adduct on the template strand in the middle of a gap and a control gap plasmid without a lesion. They transfected the plasmids into cells, recovered colonies and measured TLS by comparing the efficiency of gap repair in each. In normal cells, with Pol? present, efficient bypass occurred across the benzpyrene adduct, but in pol?-/- cells, the efficiency decreased significantly. Adding Pol? back into these mutant cells fully restored bypass efficiency of benzpyrene.

Among the postdoctoral fellows captivated by Friedberg's research findings was Visiting Fellow Wataru Nakai, Ph.D., of the Laboratory of Molecular Genetics Chromosome Stability Group.
Among the postdoctoral fellows captivated by Friedberg's research findings was Visiting Fellow Wataru Nakai, Ph.D., of the Laboratory of Molecular Genetics Chromosome Stability Group. (Photo courtesy of Steve McCaw)

Other research groups have reported that Pol? may also be involved in nucleotide excision repair and spermatogenesis. Although more work is needed to completely uncover all of Pol?'s physiological duties, Friedberg anticipated more important discoveries about this specialized polymerase. "Some of these may turn out to be very interesting," he said.


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