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Archaeal Cyclobutane Pyrimidine Dimer Class II Photolyase in Complex with UV Damaged DNA

Kelsey Dillon '14 and Jenny Shoots '14


I. Introduction

UV light can damage DNA by causing the formation of pyrimidine dimers. These dimers distort the DNA backbone, impairing replication and transcription. Photolyases are part of the DNA repair machinery of archaea, bacteria, and plants, but are not found in placental mammals. They function by breaking the cyclobutane ring of pyrimidine dimers. Photolyases function only in the presence of blue, near-UV light. They contain at least one chromophore cofactor to harvest energy from light to catalyze repair.

The structural study of photolyases to date have been of class I photolyases, found exclusively in microbes. The below tutorial is about the first crystal structure of a class II photolyase. It was isolated from the archaea Methanosarcina mazei, but multicellular eukaryotes possess class II photolyases as well. The class I, II, and III divisions were created based on sequence differences. This tutorial outlines a few of the structural differences between class I and class II photolyases. The chromophore cofactor of this photolyase, referred to as MmCPII•CPD, is FAD. Functionally, it is photoexcited to its FADH- form. It can then transfer an electron into the cyclobutane pyrimidine dimer (CPD), breaking the two carbon-carbon bonds linking the bases together.

II. Protein Structure

The structure of MmCPDII is organized in an N-terminal subdomain consisting of α helices and β sheets. The C-terminal subdomain is comprised of only alpha helices. These two subdomains are joined by a linker. The linker is very flexible, and much of its intervening structure is unknown. When the photolyase is in complex with UV-damaged DNA, it is the C-terminal domain which contacts the thymidine dimer. The cofactor FAD also makes its contacts with the C-terminal domain.

III. FAD Binding Site

Photolyases require a chromophore cofactor, in this case FAD, for both recogniction of CPD lesions and for catalysis. Upon excitation by light, photolyases reduce their cofactor FAD into FADH-. This activity requires a photoreduction pathway which transfers excited electrons to FAD, shown here in its oxidized form. Class I photolyases depend on three linear tryptophan residues for electron transfer. The class II photolyase shown here possesses several tryptophan and tyrosine residues which can transfer electrons to FAD , but photoreduction has been shown to be dependent only upon two tryptophans. Electrons are transferred from W360 to W381 to FAD. Additionally, the asparagine residue N403 stabilizes FAD during photoreduction.

IV. Interactions with Pyrimidine Dimer

In the MmCPDII•CPD-DNA complex, the DNA has a synthetic CPD (cyclobutane pyrimidine dimer) lesion in its central position. The dsDNA adopts a typical B-type conformation, but is severely kinked at the lesion site. The CPD lesion of the kinked dsDNA is flipped out of the duplex into the active site of MmCPDII and bound there.

In MmCPDII, conserved tryptophans W305 and W421 form the L-shaped walling of the active site that clamps the CPD lesion together with the side chain of the conserved M379.

During repair a transient radical form of the CPD lesion forms once FADH- transfers an electron to the lesion. E301 stabilizes the CPD radical anion by proton transfer after the breaking of the thymine dimer. It forms hydrogen bonds with the 5’-thymine of the lesion.

A cluster of six water molecules occupies the space between the CPD lesion and the active site’s walling.

One of these water molecules replaces the conserved side chain asparagine found in class I photolyases (replaced by a glycine in MmCPDII) by taking over the bridging interaction between the C4-carbonyl group of the 3’-thymine and the C2’-hydroxl of the FAD’s ribityl group.

The other five water molecules are located as a square-pyrimidal cluster between the 3’-thymine and the diphosphate group of FAD.

V. References

Kim, S.-T. and A. Sancar 1993. Photochemistry, Photophysics, and Mechanism of Pyrimidine Dimer Repair by DNA Photolyase. Photochemistry and Photobiology, 57: 895–904.

Kiotntke, S., Geisselbrecht, Y., Pokorny, R. Carell, T., Batschaer, A., and L.-O. Essen. 2011. Crystal structures of an archael class II DNA photolyase and its complex with UV-damaged duplex DNA. EMBO Jour. 30: 4437-4449.

Malhotra, K., Kim, S.-T., Walsh, C., and A. Sancar. 1992. Roles of FAD and 8-Hydroxy-5-deazaflavin Chromophores in Photoreactivation of by Anacystis nidulans DNA photolyase. J. Biol Chem. 267(22): 15406-15411.

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