Uracil-DNA Glycosylase

Rei Mitsuyama '15 and Holden Richards '15

Contents:

I. Introduction

Removal and replacement of the altered bases is the most common method cells employ to rid their DNA of damaged bases. Base excision reapir (BER) and nucleotide excision repair (NER) are the two principle repair pathways. The first enzyme in the BER process is glycosylase. Uracil DNA glycosylase (UDG) is one of 11 different human DNA glycosylases that have been identified5.

Though functionally only present in RNA, uracil is occasionally misincorporated into DNA. This most commonly is a result of cytosine deamination. UDG scans the minor groove for damage and once it finds the error, it hydrolyzes the glycosidic bond to release the base from the sugar-phosphate backbone. Remarkabley, it is able to all of this without the aid of other cofactors. It then leaves the AP site (apyrimidinic site) open for an AP endonuclease to cut the backbone at the 5' end of the AP site. This marks the initiation of this BER5. 

II. General Structure

UDG is a composed of four parallel beta sheets surrounded by eight alpha helices in the following order: α1, α2, α3, β1, α4, α5, β2, α6, β3, α7, β4, α81. The active site is comprised of . The Leu272 loop is involved in base stacking interactions with the DNA. Along with the 4-Pro loop and the Gly-Ser loop , it compresses backbone phosphates, bending the DNA. The comprised of residues Tyr275 and Arg276 are also part of the Leu272 Loop. The uracil specificity region is responsible for recognition of the correct flipped-out base while the water-activating loop is vital in catalysis3.  

III. DNA Binding

Uracil-DNA glycosylase is lesion-specific (as the name suggests) for in DNA. For DNA to be bound by UDG, it must be in the B form. This makes sense because UDG acts on newly synthesized DNA containing a misincorporated base (uracil). The exact mechanism by which UDG scans the DNA remains unknown. However, it seems to involve the serine residues of - the Leu272 loop, 4-Pro loop, and Gly-Ser loop. The damaged base is flipped out, sitting in the specificity pocket of the glycosylase, which makes it project away from the double helix. These Ser residues form with the phosphates both 5' and 3' to the uracil. This interaction compresses the DNA backbone, bringing the UDG into closer proximity of the uracil, aiding its recognition. Initial detection of uracil misincorporation by backbone compression is coupled to the minor groove reading head residues Tyr275 and Arg276, which make hydrogen bonds to the uracil N3. The closest interaction between the enzyme and the uracil involves two with the Asn204 of the uracil specificity region3. This hydrogen bonding is thought to be the strongest and most important in recognizing the uracil lesion. It is believed that these hydrogen bonds allow UDG to distinguish between cytosine and uracil4. The total enzyme-DNA interface is small and most of the interactions involve this flipped-out base.

IV. Enzymatic Activity

The UDG (~27 angstroms long between Ser247 and Val164 before binding) has a conical shape that can accommodate a DNA double helix at its wide end, which is about 21 angstroms (before binding) between His212 and Leu272, but not at its narrow end, which is only 10 angstroms between Pro150 and Pro165. This suggests that the groove floor cannot directly bind dsDNA without significant conformational change. However, no conformational change has been observed, suggesting that DNA only binds at the wide end of the enzyme's active site4.

In order for the misincoprporated uracil to be situated in the active site, it must be into an extrahelical conformation. It was hypothesized that this was caused by a "push" from residue emanating from the Leu272 loop, between beta sheet 4 and alpha helix 8, as it penetrates the DNA through the minor groove and replaces the flipped-out uracil2. However, the crystal structures have confirmed that this "push" is not essential for base-flipping, as an extrahelical uracil has been observed in crystal structures without the presence of Leu272 within the DNA double helix. Nevertheless, the "push" has been linked to enhanced efficiency of excision3. This can be seen as a consequence of two major actions of Leu272. The insertion of Leu 272 into the double helix stabilizes the extrahelical-uracil conformation by forming new interactions, allowing this conformation to be favorable. It also aids the formation of the recognition pocket as the insertion brings conserved residue His268 close enough to with the uracil O23.

The ultimate reaction catalyzed by UDG cleavage of the glycosidic bond between the uracil base and its corresponding deoxyribose sugar, releasing uracil from the DNA backbone. seems to be chiefly responsible, through its interaction with water. Once UDG binds DNA, the His148 rotates into a conformation that allows it to deprotonate the catalytic molecule. This activates the water molecule by giving it a negative charge and thereby making it a strong nucleophile. Then, by nucleophilic attack, the water molecule breaks the glycosydic bond, freeing the uracil base. To aid in this process, Asp145 hydrogen bonds with N3 of the base, polarizing the glycosidic bond, which makes it more susceptible to this attack1.

V. References

(1) Mol, Clifford D., Andrew S. Arvai, Geir Slupphaug, Bodil Kavli, Ingrun Alseth, Hans Krokan, and John A. Tainer. 1989. Crystal structure and mutational analysis of human uracil-DNA glycosylase: Structural basis for specificity and catalysis. Cell Press 80:869-878.

(2) Mol, Clifford D., Andrew S. Arvai, Russell J. Sanderson, Geir Slupphaug, Bodil Kavil, Dale W. Mosbaugh, and John A. Tainer. 1995. Crystal Structure of Human Uracil-DNA Glycosylase in Complex with a Protein Inhibitor: Protein Mimicry of DNA. Cell Press 82:701-708.

(3) Parikh, Sudip S., Clifford D. Mol, Geir Slupphaug, Sangeeta Bharati, Hans E. Krokan, and John A. Tainer. 1998. Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase of DNA. The EMBO Journal 17:5214-5226.

(4) Pearl, Laurence H. 1987. Structure and function in the uracil-DNA glycosylase superfamily. Elsevier 460:165-181.

(5) Watson, James D. Alexander Gann, Tania A. Baker, Michael Levine, Stephen P. Bell, and Richard Losick. 2014. Molecular Biology of the Gene. Cold Spring Harbor Laboratory Press 7:326-329.

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