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New DNA glycosylases from the helix--two-turn--helix superfamily:
Structures and biochemical characterization
Yudkina A.V.
1,2
, Naumenko M.B.
2
, Garcia-Diaz M.
3
, Zharkov D.O.
1,2
1
SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia 
2
Novosibirsk State University, Novosibirsk, Russia 
3
Stony Brook University, Stony Brook, NY, USA
DNA glycosylases are enzymes that maintain the genome integrity, representing a key 
element of the base excision DNA repair system. Due to their ability to recognize base lesions 
created by deamination, ionizing radiation, alkylating and oxidizing agents, these enzymes 
are still a subject of acute interest. DNA glycosylases are divided into several unrelated struc-
tural superfamilies, one of which, the helix–two-turn–helix (H2TH) superfamily, consists 
of enzymes acting on a variety of oxidized purines and pyrimidines. Eight families within 
the H2TH superfamily were known until recently: Fpg, Nei, NEIL1, NEIL3, NEIL3, MMH, 
AcNei1 and AcNei2, named after their representative members that had been extensively 
studied. Recently, whole-genome sequencing revealed four groups of new H2TH members 
present in some bacterial species. Importantly, these new enzymes show differences from 
the existing representatives of the H2TH superfamily in several critical regions responsible 
for DNA binding, damaged base eversion and recognition. In this work we have cloned and 
investigated two such enzymes, which we term Flp (Fpg-Like Proteins): Flp1 from 
Strepto-
myces coelicolor 
and Flp3b from 
Bacteroides thetaiotaomicron
. We have used an optimized 
protocol of protein expression and purification to obtain these enzymes in an active form. Both 
enzymes preferentially cleaved DNA containing oxidized pyrimidines (5-hydroxyuracil and 
dihydrouracil) and abasic sites, and were unable to remove oxidized purines (8-oxoguanine, 
8-oxoadenine), deaminated bases (uracil, hypoxanthine), and epigenetically modified bases 
(5-methylcytosine, 5-hydroxymethylcytosine). Both Flp1 and Flp3b, unlike many other DNA 
glycosylases, were also active on single-stranded DNA, and had no opposite-base preference. 
We have crystallized and solved the structure of Flp3b at 2.0 Å resolution. It had an atypical 
N-terminal catalytic residue (Lys instead of Pro in most other H2TH enzymes) and demon-
strated significant deviation from known structures of Fpg, Nei, and NEIL1 in several loops 
that specifically interact with damaged DNA. Overall, Flp proteins appear to be redundant for 
oxidative damage repair, since the respective genomes also encode “typical” H2TH enzymes. 
We speculate that, despite their ability to excise damaged pyrimidines, Flp proteins may have 
evolved in response to unique genotoxic challenges, such as DNA-damaging secondary metab-
olites, faced by some bacteria in their native environment.
This study was supported by RFBR (grant 17-04-01761-a).

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Всероссийская мультиконференция с международным участием «Биотехнология – медицине будущего»
29 июня - 2 июля 2019 г., г. Новосибирск, Россия
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