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Ultraviolet Damage Endonuclease

Q Tashiro '15 and Rachel Rhee '15


Contents:


I. Introduction


    Cells have developed a range of repairing mechanisms in order to guard genetic integrity against normal metabolic activities and environmental factors such as UV light and radiation. Such damage can form molecular lesions that cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene the DNA encodes3. Thus, the cell's DNA repair system is constantly active as it responds to damage in the DNA structure. DNA is most commonly cleansed of damaged bases by repair systems that remove and replace the altered bases including direct reversal, nucleotide excision repair (NER), base excision repair (BER), and ultraviolet damage endonuclease repair (UVDE). The ultraviolet damage endonuclease repair system distinguishes itself by recognizing and subsequently nicking DNA containing different types of damage with the single, multifunctional UVDE enzyme2. The UVDE repair system involves an endonuclease activity to catalyze the hydrolysis of ester linkages within nucleic acids by creating internal breaks and a metal ion binding site that interacts selectively and non-covalently with metal ions within the active site2
    UV light induces damage in the cell resulting in two major types of photodamage including cyclobutane pyrimidine dimers (CPDs) and photoproducts (PPs) that will eventually lead to cytotoxic and mutagenic damage2. Cellular DNA have developed repair mechanisms that are able to restore photoproducts bases back to their original undamaged states. Specifically, the UVDE of Thermus thermophilus is thought to initiate the first step in an alternative excision repair pathway for the removal of UV light-induced DNA damage. The UVDE functions as a broad specificity, ATP-independent 5’ endonuclease for initiating the repair of DNA lesions, CTDs, and UV photoproducts3. The enzyme performs the initial step in an alternative excision repair pathway by cleaving the phosphodiester backbone of substrates containing both CPDs and PPs immediately adjacent to the 5’ phosphate of the damaged nucleotides2.  
   
    Most proteins involved in DNA repair have very narrow specificity for addressing only one damage. However, the UVDE is only a single protein that has a relatively broad substrate specificity, which makes it an interesting model to study DNA repair.
The closest homolog of the UVDE is Endo IV and while they share major structural features, the UVDE is much more versatile in its ability to repair DNA damage. Preliminary work has shown that the structural flexibility from the more substantial rearrangements of UVDE upon DNA binding might explain its broader substrate specificity compared to that of Endo IV2.


II. General Structures

    The general structure of the UVDE protein includes a single-domain TIM barrel (lacking the α8 helix) of the prototypical TIM-barrel fold (Paspaleva). The TIM barrel is a conserved protein fold consisting of 8 α-helices and 8 parallel β- strands  and is considered one of the most common protein folds4. The UVDE is classified as a member of the TIM-barrel family of divalent metal-dependent enzymes due to the three anomalously scattering metal ions , located closely to the C terminus and due to the close proximity of the protein’s N and C termini2.     The UVDE structure reveals a novel use of the TIM barrel fold for binding DNA for damage recognition and catalysis. The enzyme must bind and scan normal DNA via electrostatic complementarity and hydrogen bonding to the DNA phosphate backbone from β barrel loops and α-helical dipoles identically positioned by the α8β8 framework1. DNA damage detection proceeds by insertion of side chains from minor groove recognition loops to provide DNA backbone compression and flipping of the target AP site and its opposing nucleotide out of the helix1.  



III. Base Flipping


    Two residues that stick out (Gln 104 and Tyr 105) are called the probing finger and are responsible for probing the DNA for damage. These residues function in close proximity of the metal coordination site and present the scissile phosphodiester bond at the 5` side of the lesion to the active site2. A small rearrangement of two residues at its C terminus, Lys-273 and Glu-274, create a pocket to fit both the damaged base and the flipped-out base opposing this damage


IV. DNA Binding

DNA Binding site     A distinct crescent-shaped groove formed by the C-terminal end of the TIM barrel forms the enzyme active site. The wide groove (29 ) which contains extensive positive charges on both sides and three active site ions impart an overall net positive electrostatic potential throughout the groove that complements the negatively charged DNA phosphate backbone. This pocket allows the UVDE to comfortably harbor the kinked duplex.

    At the bottom of the DNA binding groove there is a loop, formed from the previously mentioned probing fingers, Gln 104 and Tyr 105, that also serve to stabilize the kink in the DNA duplex at the position of damage. This overall structure demonstrates how the TIM barrel fold can be optimized for DNA binding, therefore is a fold that recurs in other proteins that bind to, modify, or repair nucleic acid substrates.

    A homolog of UVDE, Endo IV, is shown bound to a damaged DNA. The binding to dsDNA is mediated via five DNA recognition loops (R loops) that emanate from the positively charged C-terminal end of the central β barrel. The position of the DNA against the protein shows how it fits along the pocket of the DNA.

    Significant side chain rearrangements of 
β2–α2 residues Asn-35, Gln-36, Arg-37, Gln-38, and Trp-39 pack against the hydrophobic carbon atoms of the minor groove atoms to stabilize the extrahelical nucleotides and the 90 bend in the DNA 2. The enzyme facilitates contacts via electrostatic complementarity and hydrogen bonding to contacts anchor the flipped-out abasic nucleotide to the enzyme active site and position its 5` phosphate such that it is intimately ligated to all three ions1. The rigid TIM barrel framework positions these five R loops to provide extensive charge, chemical, and shape complementarity for the entire interface 2.
    



V. Role of Metal Ions in the Active Site

    Metal ions that are present within close proximity of the protein’s N and C termini, classifies UVDE as a member of the TIM-barrel family of divalent metal-dependent enzymes2. The first metal ion is octahedrally coordinated by the side chain interactions between Glu-175, Glu-269, His-231, and Asp-200 residues and two oxygen atoms from a phosphate ion. The second metal ion forms a distorted bipyramidal coordination by His-101, His-143, and Glu-175, along with two oxygen atoms from the phosphate ion. The third metal ion has an irregular four-fold coordination by His-244, His-203, one water molecule and one oxygen atom from the phosphate ion.

    Studies show that the UVDE active site metal ions are likely to be directly involved in phosphodiester cleavage, also observed in other DNA repair enzymes such as Endo IV. These positively charged metal ions can act as a Lewis acid to stabilize a water-derived hydroxide attacking the phosphodiester backbone of DNA which will counteract the developing negative charge on the DNA during cleavage reaction2. T. thermophilus UVDE (Tth-UVDE) shows high incision on substrates containing CPD or 6-4PP however incision of the abasic sites is much less efficient2.

    For the UVDE to incise DNA containing CPD and 6-4PP UV lesions, Mn2+ is required for the UVDE catalytic activity. Ongoing studies have shown that the specific divalent metals present in the active site vary depending on other UVDE homologs. For instance, the S. pompe protein can only utilize Mg2+ as a catalytic cofactor2 however, T. thermophilus UVDE is not able to stimulate enzymatic activity with Mg2+.

3...2...1!


VI. References

1. Hosfield DJ, Guan Y, Haas BJ, Cunningham RP, Tainer JA. Structure of the DNA repair enzyme endonuclease IV and its DNA complex: Double-nucleotide flipping at abasic sites and three-metal-ion catalysis. Cell. 1999;98(3):397-408.

2. Paspaleva K, Thomassen E, Pannu NS, Iwai S, Moolenaar GF, Goosen N, Abrahams JP. Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease. 2007;15:1316-1324.

3. Siede W, Doetsch PW, Wah Kow Y, Doetsc PW.
DNA damage recognition. 1st ed. New York: Taylor & Francis Group; 2005.http://books.google.com/books?hl=en&lr=&id=JB6OKFsBjNQC&oi=fnd&pg=PR3&dq=UVDE+protein&ots=li1I1Xqj0B&sig=8jdsETEJhNhoQTvle
7aQrM7AewI#v=onepage&q=UVDE%20protein&f=false.

4. Wierenga RK. The TIM-barrel fold: A versatile framework for efficient enzymes. FEBS Lett. 2001;492(3):193-198.

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