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Biological Sciences Division
Research Highlights

June 2005

NMR study of DNA repair protein dynamics featured on journal covers

A study supported by the U.S. Department of Energy's Office of Biological and Environmental Research Program was recently featured on the cover of three consecutive issues of the journal DNA Repair. The cover graphic, which summarizes dynamics and surficial modifications of a DNA repair protein upon binding damaged DNA, accompanied an article entitled "Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): A dynamic description of the DNA/protein interface." The authors are PNNL scientists Garry W. Buchko and Michael A. Kennedy, Washington State University scientist Kathleen McAteer, and University of Vermont professor Susan S. Wallace.

The propagation of life depends on the fidelity of the hereditary molecule DNA. However, DNA is a reactive molecule, and in aqueous solution it interacts with many types of exogenous and endogenous agents of which the most important are reactive oxygen species, such as hydrogen peroxide, superoxide radical anions, and hydroxyl radicals. Such reactions can result in the generation of many types of DNA damage in the course of normal metabolism.

More than 100 different DNA modifications have been identified from the reaction of DNA with endogenous reactive oxygen species. One of the most prominent lesions generated is 7,8-dihydro-8-oxoguanine, which is implicated in mutagenesis, carcinogenesis, and aging. While a large number of chemically and physically distinct DNA lesions are generated by reactive oxygen species, base excision repair (BER)—the mechanism that repairs most of these lesions—is composed of a relatively small number of DNA glycosylases that aid in DNA repair. In human cells, ~10 of these enzymes involved in BER have been identified, while eight have been found in the bacterium Escherichia coli. One of these in E. coli is formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa.

X-ray analysis of 3D crystal structures of Fpg, covalently and non-covalently bound to DNA, revealed many of the functionally important residues that participate in DNA binding and enzyme catalysis. However, crystal structures present a static snapshot of molecules and molecular complexes. In the real biological environment, molecules are moving around, and consequently, crystal studies fail to shed light on the dynamic nature of the repair process. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, the high-field NMR spectrometers at the Environmental and Molecular Sciences Laboratory (http://www.emsl.pnl.gov/) were used to study E. coli Fpg free and bound to a double-stranded DNA oligomer (13-PD) containing propanediol, a non-hydrolyzable abasic-site analogue. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate time-scale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. All other residues did, however, exhibit tight DNA binding characteristic of slow exchange.


Figure 1. Mapping the dynamics at the DNA/Protein interface of the Escherichia coli Base Excision Repair Enzyme Formamidopyrimidine-DNA Glycosylase (Fpg). Left) Molscript ribbon representation of Ec-Fpg in the Ec-Fpg/DNA covalently cross-linked crystal structure (1K82). Right) A CPK-space filled model of the Ec-Fpg/DNA complex. The Ec-Fpg residues are colored to reflect the backbone dynamics of Ec-Fpg in solution before and after non-covalent association with a DNA substrate, 13-PD, a 13-residue double-stranded DNA oligomer containing 1,3-propanediol, a non-hydrolyzable abasic-site analogue. Full Image

As illustrated on the journal covers (Figure 1), novel CPMG-HSQC NMR experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that was quenched upon binding 13-PD. Collectively, these observations reveal that, in solution, Fpg contains flexible regions, including the DNA binding site, and that DNA binding is associated with changes in local dynamics, and perhaps structure, that are propagated to distant parts of the protein. The dynamic nature of Fpg, especially at the DNA binding surface, may be key to its processive search for DNA damage.

References

Buchko GW, K McAteer, SS Wallace, and MA Kennedy. 2005. "Solution-State NMR Investigation of DNA Binding Interactions in Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg): A Dynamic Description of the DNA/Protein Interface." DNA Repair 4(3):327-339. DOI:10.1016/j.dnarep.2004.09.012. 

Buchko GW, SS Wallace, and MA Kennedy. 2002. "Base Excision Repair: NMR Backbone Assignments of Escherichia coli Formamidopyrimidine-DNA Glycosylase." Journal of Biomolecular NMR 22(3):301-302. DOI:10.1023/A:1014903518628.

Buchko GW, NJ Hess, V Bandaru, SS Wallace, and MA Kennedy. 2000. "Spectroscopic Studies of zinc (II) and Cobalt (II) Associated Escherichia coli Formamidopyrimidine-DNA Glycosylase: Extended X-Ray Absorption Fine Structure Evidence for a Metal Binding Domain." Biochemistry 39(40):12441-12449. DOI:10.1021/bi001377k.


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