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Dissecting the Complexities of NHEJ in DNA Repair

By Robin Arnette
October 2007

Robert London, Ph.D., Lars Pedersen, Ph.D., and Thomas Kunkel, Ph.D., members of the NIEHS Laboratory of Structural Biology, collaborated with Ramsden to generate a computer model of the amino acid in loop 1 of pol ? (R175) that interacts with DNA.
Robert London, Ph.D., Lars Pedersen, Ph.D., and Thomas Kunkel, Ph.D., members of the NIEHS Laboratory of Structural Biology, collaborated with Ramsden to generate a computer model of the amino acid in loop 1 of pol ? (R175) that interacts with DNA. (Graphic courtesy of Dale Ramsden)

Ramsden used site-directed mutagenesis to change the arginine in pol ? to an alanine (R175A). Both in vitro and in vivo results indicated that the mutant pol ? is deficient in NHEJ and therefore requires a DNA template when promoting end joining
Ramsden used site-directed mutagenesis to change the arginine in pol ? to an alanine (R175A). Both in vitro and in vivo results indicated that the mutant pol ? is deficient in NHEJ and therefore requires a DNA template when promoting end joining. (Graphic courtesy of Dale Ramsden)

During a visit to NIEHS on September 12, Dale Ramsden, Ph.D. (http://www.med.unc.edu/biochem/ramsden) Exit NIEHS, associate professor in the Department of Biochemistry and Biophysics and the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill School of Medicine, spoke to a standing-room only audience in F193 in the Rall building. His seminar, "Nonhomologous End Joining and Base Excision Repair: Brothers from Another Mother," discussed some of the findings of his work. Robert London, Ph.D., head of the Nuclear Magnetic Resonance Group in the NIEHS Laboratory of Structural Biology, hosted the event.

Ramsden began his seminar with an overview of DNA repair. During a single strand break, several mechanisms, such as nucleotide excision repair and base excision repair, lead to accurate repair of the damage. "But when you have a double-strand break caused by ionizing radiation, there are actually two ways to fix it: homologous recombination (HR) or nonhomologous end joining (NHEJ)," Ramsden explained. In HR the ligation of broken ends requires a homologous sequence or template to guide repair, but in NHEJ no such template is needed.

Ramsden went on to explain that in eukaryotes several core factors are required for NHEJ. One of the factors, DNA Ku protein, exists as a heterodimer of Ku70 and Ku80 and recruits the catalytic subunit of DNA protein kinase (DNA-PKcs). DNA ligase IV and XRCC4 are also required, but Ramsden found in his studies that the protein XLF, also known as Cernunnos, promoted synaptic maintenance and allowed the Ku-XRCC4-ligase IV complex to join mismatched ends directly.

During the second portion of the talk Ramsden discussed the interactions between DNA polymerases-deoxynucleotide transferase (TdT), pol ? and pol ?-in NHEJ. He knew from research done in other labs that TdT was template-independent, but he wanted to know if that held true for pol ? and pol ?. Using an in vitro assay Ramsden measured the activity of pol ? and pol ? by adding them separately into solution that either contained or lacked the Ku-XRCC4-ligase IV template. In the presence of the template both polymerases ligated the ends and extended the primer, but in the absence of template, pol ? lacked activity. This suggested that pol ? was template-independent like TdT.

To find out what part of pol ? was involved in binding and extending DNA, he turned to structural data. Ramsden's NIEHS collaborators, including London, Lars Pedersen, Ph.D. and Thomas Kunkel, Ph.D., generated computer models and more recently a structure that not only indicated that loop 1 in pol ? had a similar structure to loop 1 in TdT, pol? and pol ?, but they also identified additional requirements for pol ?'s activity. This included an arginine at amino acid position 175 (pol ? R175) which interacts with the end containing DNA template. Ramsden used site-directed mutagenesis to change the arginine to alanine (pol ? R175A). It turned out that although the mutant pol ? R175A has wild type TdT-like template-independent activity, it is template-dependent when it comes to promoting end joining.

Ramsden's research into determining the exact mechanism and the players that are involved in NHEJ are important because cells suffer double-strand breaks on an almost daily basis. If this damage isn't fixed, it can lead to cell death or cancer.

The one take home message Ramsden wanted to convey was simple. "The pathway that I work on [non-homologous end joining] is far more sophisticated in making accurate junctions than we would have expected," he said. "We commonly think of this as an error-prone pathway, but it is a lot more precise than it looked initially."


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