Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56

Abstract

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.

Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones

“…However, inhibition of SbcCD is unlikely to contribute to the antibacterial potentiation effect observed here, because cells lacking SbcCD activity are not sensitive to ciprofloxacin (Henderson and Kreuzer, 2015; Aedo and Tse-Dinh, 2013; Liu et al, 2010). DNA mimicry has been observed previously for phage-encoded proteins that target type I restriction endonucleases and glycosylases (Kennaway et al, 2009; Baños-Sanz et al, 2013; Cole et al, 2013) and it may be a common mechanism for bacteriophages to modulate DNA replication and repair in their hosts. Bacteriophage P22 codes for another, distinctive RecBCD inhibitor called Abc2 but this operates by a poorly-characterised mechanism (Murphy, 2000).…”

Global Transcriptional Analysis of Virus-Host Interactions between Phage ϕ29 and Bacillus subtilis

“…If uracil residues appear in ssDNA regions of replicative intermediates, the action of host uracil DNA-glycosylase (UDG) will introduce a nick into the phosphodiester backbone producing the loss of the terminal region. To avoid this process, phage 29 encodes a UDG inhibitor called p56 (57,58).…”

A robust photoluminescence screening assay identifies uracil-DNA glycosylase inhibitors against prostate cancer

“…14 The uracil-DNA glycosylase inhibitor (UDGI), produced in of Bacillus subtilis bacteriophage PBS1, is a $9.5 kDa protein that is used in the literature as a model inhibitor of UDG. 15 Other inhibitors of UDG have been reported, such as SSP0047, p56, and uracil aldehyde small molecules, 13,[16][17][18][19][20] however none of these have undergone further indepth disease application research. The discovery of new inhibitors of UDG and the development of methods for their identication could offer the potential for synergistic therapeutic strategies with 5-FU against cancer, including prostate cancer.…”

“…21 Other reported methods for identifying UDG inhibitors include fragment-substrate tethering, bioinformatics, radioisotopic labeling, chemical cross-linking and affinity chromatography techniques. 13,[16][17][18][19][20] However, these methods tend to be time-consuming, unwieldy and/or may necessitate stringent safety measures to control radiographic exposure. 22 Therefore, new in vitro strategies for the rapid and efficient screening of UDG inhibitors are still desired.…”