SMUG2 DNA glycosylase from Pedobacter heparinus as a new subfamily of the UDG superfamily

Abstract

Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are essential to maintain genome integrity, in which the base excision repair (BER) pathway plays a major role in the removal of base damage. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for excising the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Using bioinformatics tools, we identified a family 3 SMUG1-like DNA glycoyslase from (named Phe SMUG2), which displays catalytic activities towards DNA containing uracil or hypoxanthine/xanthine. Phylogenetic analyses show that SMUG2 enzymes are closely related to family 3 SMUG1s but belong to a distinct branch of the family. The high-resolution crystal structure of the apoenzyme reveals that the general fold of Phe SMUG2 resembles SMUG1s, yet with several distinct local structural differences. Mutational studies, coupled with structural modeling, identified several important amino acid residues for glycosylase activity. Substitution of G65 with a tyrosine results in loss of all glycosylase activity. The crystal structure of the G65Y mutant suggests a potential misalignment at the active site due to the mutation. The relationship between the new subfamily and other families in the UDG superfamily is discussed. The present study provides new mechanistic insight into the molecular mechanism of the UDG superfamily.

An unconventional family 1 uracil DNA glycosylase in Nitratifractor salsuginis

“…We have encountered a similar scenario during our structural characterization on Phe SMUG2. When we introduced the G65Y mutation, the resulting structure of the mutant is highly similar to that of the WT enzyme .…”

Identification of a prototypical single-stranded uracil DNA glycosylase from Listeria innocua

“…Amplification of mutant DNA and DpnI mediated site-directed mutagenesis procedures were carried out as previously described with modification by using primers carrying the desired mutations [32, 33]. To express the C-terminal His-6-tagged wild-type and mutant Lin SMUG1-like glycosylase gene, the recombinant plasmids were transformed into E. coli strain BL21 (DE3 Δ slyD Δ mug Δ udg Δ nfi Δ nth Δ ndk ) by electroporation.…”

Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases

“…Some of them, such as Family 1, where extensively studied human and E. coli enzymes belong, and Family 4, initially discovered in extremophilic archaea and bacteria and containing an FeS cluster, seem to be bona fide uracil–DNA glycosylases with little activity on other substrates. Others, such as SMUG1 (Family 3), FeS-containing Family 5, and the recently discovered SMUG2 and Bradyrhizobium diazoefficiens uracil–DNA glycosylase (BdiUng)-like enzymes, have wider substrate specificity that may additionally include other U derivatives (5OHU, 5-hydroxymethyluracil, 5-formyluracil), Hx, and Xan [ 116 , 117 , 118 , 119 ]. Thymine–DNA glycosylase (TDG) present in animals and fungi is involved in active epigenetic demethylation, removing oxidized and/or deaminated derivatives of 5-methylcytosine [ 120 , 121 ], and together with its bacterial homolog Mug may be the primary glycosylase for exocyclic pyrimidine adducts such as 3, N 4 -methylcytosine, or for 7,8-dihydro-8-oxoadenine (8oxoA) [ 122 , 123 ].…”