Viruses with U-DNA: New Avenues for Biotechnology

Abstract

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA-protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes.

Keywords: biotechnology; phages; uracil-DNA.

Figure 1

Pyrimidine nucleotide metabolism.

Figure 2

Structure of the UNG protein bound to DNA containing a substrate analogue. On the left: UNG enzyme protein (green ribbon model) binds DNA (orange ribbon model) containing the 2′-deoxy-pseudouridine-5′-monophosphate substrate analogue (stick model, atomic coloring: O: red, N: blue, P, C: orange) (PDB code: 1EMH). On the right: close-up of amino acids (stick model, atomic coloring: O: red, N: blue, C: green) which directly interact with the uracil base (stick model, atomic coloring: O: red, N: blue, P, C: orange).

Figure 3

Multiple sequence alignments of three uracil-DNA glycosylase inhibitor proteins. ‘*’ (asterisk) indicates positions which have a single, fully conserved residue; ‘:’ (colon) indicates conservation between groups of strongly similar amino acids; ‘.’ (dot) indicates conservation between groups of weakly similar amino acids.

Figure 4

A structural comparison of UGI, SAUGI and p56 and their complexes with UNG. (A) Three-dimensional structures of the three known uracil-DNA glycosylase inhibitors, depicted as ribbon models (in green). The two monomers of p56 are shown in different colors (green and cyane). (B) Electrostatic representation of three inhibitor proteins (surface models colored according to electrostatics - red symbolizes negative charge, blue positive and white is neutral) in their complexes with UNG (ribbon model). It is clearly visible that in all three cases, the inhibitor protein binds to the UNG DNA-binding groove, mimicking the negative charge of the DNA. The figures were produced using the PyMOL program based on structural data from PDB database (PDB ID: UGI: 1UGI; SaUGI: 3WDG; p56: 2LE2; E. coli UNG complex with UGI: 1LQM; S. aureus UNG complex with SaUGI: 3WDG; B. subtilis UNG complex with p56 dimer: 3ZOQ) [45,54,56,57,58].