UDG

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Introduction

Nearly all organisms employ DNA repair pathways to correct damage in DNA molecules. DNA damage can be caused by endogenous (e.g., DNA polymerase infidelity, reactive oxygen species) and/or exogenous substances (e.g., UV radiation, X-rays). Bulky DNA lesions caused by certain environmental mutagens, such as thymine dimers or 6,4-photoproducts, can eventually be repaired by the nucleotide excision repair (NER) pathway. NER provides a necessary defense mechanism for a large number of organisms. Repair of cyclobutane thymine dimers and 6,4-photoproducts in prokaryotes can also be mediated by photolyases, a process that relies on the non-covalently bound cofactor flavin adenine dinucleotide. NER in eukaryotes involves more major proteins, including XPA, XPB, XPC, XPD, XPE, XPF, XPG, CSA, and CSB, and many additional proteins, including ERCC1, RPA, RAD23A, and RAD23B.

Modifications of DNA bases through oxidation, deamination, or alkylation can be repaired by the base excision repair (BER) pathway. BER is initiated by DNA glycosylases responsible for the removal of cytotoxic and mutagenic bases from DNA. DNA glycosylase consists of monofunctional DNA glycosylases and bifunctional DNA glycosylases/AP lyases. Water is used as a nucleophile to cleave the N-glycosidic bond between the target base and deoxyribose in monofunctional enzymes, thereby releasing a free base and creating an apurine/apyrimidine (AP) site (also called abasic site). The bifunctional DNA glycosylases/AP lyases use an amino group of the enzyme as a nucleophile to form a Schiff’s base intermediate.

Figure 1. Types of lesions found in DNA (Schormann, N.; et al. 2014)

Uracil-DNA glycosylase families - Overview

Uracil-DNA glycosylases (UDGs) are monofunctional glycosylase enzymes responsible for the removal of uracil from DNA. Spontaneous deamination of cytosine (Cyt) or incorporation of dUMP during DNA synthesis may lead to the production of uracil (Ura) in DNA. UDG enzymes have been identified in a large number of organisms, including archaea (e.g., Sulfolobus solfataricus, Methanococcus jannaschii), eubacteria (e.g., Escherichia coli, Bacillus subtilis), eukaryotes (e.g., Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans) and large DNA viruses ( herpes virus, poxvirus).

UDGs can be divided into six families based on substrate specificity. A sequence alignment of the characteristic motifs in the six UDG families is shown in Figure 2, underscoring the clear separation between the six families. In groups I-V, only the N- and C-terminal motifs corresponding to the extended regions of the "water-activated" loop and the "Leu-inserted" loop are highlighted. In family VI, the extended region of the "water-activated loop" in the helix-hairpin-helix (HhH) motif is shown together with the [4Fe-4S] cluster binding motif. For family VI, the extended region of the "water-activated loop" in the helix-hairpin-helix (HhH) motif is shown together with the [4Fe-4S] cluster binding motif.

Figure 2. Phylogenetic tree of sequence alignment (Schormann, N.; et al. 2014)

Family I UDGs (also known as UNGs), E. coli, and human UNG proteins are the most widely studied enzymes in the UDG superfamily. UNGs are highly specific for uracil (Ura) and do not excise 5-substituted Ura. Family I UDGs are primarily responsible for the removal of uracils from DNA resulting from cytosine deamination. Currently, approximately 75% of all UDG structures preserved in protein databases are family I enzymes.

Representative enzymes of family II UDGs include E. coli mispair-specific uracil glycosylase (MUG) and human thymine DNA glycosylase (TDG), both of which are mismatch-specific enzymes for dsDNA. Studies have shown that MUG can also excise Thy from T:G mispairs at high enzyme concentrations.

Family III UDGs (sMUGs) are enzymes that exist only in higher eukaryotes. sMUG enzymes can not only use dsDNA as a substrate, but also have high affinity for uracil-containing dsDNA substrates. In addition to this, sMUG enzymes can also excise 5-hydroxymethyluracil (5-HmU).

The remaining three families of UDGs exist in thermophilic and hyperthermophilic eubacteria and archaea. These enzymes all have four conserved cysteine residues that act as ligands for four Fe atoms in a cubic iron-sulfur (4Fe-4S) cluster. UDG enzymes in these families are responsible for the removal of Ura from uracil-mismatched dsDNA. Of these, families IV and VI also excise Ura from ssDNA substrates.