Life without dUTPase

Abstract

Fine-tuned regulation of the cellular nucleotide pools is indispensable for faithful replication of Deoxyribonucleic Acid (DNA). The genetic information is also safeguarded by DNA damage recognition and repair processes. Uracil is one of the most frequently occurring erroneous bases in DNA; it can arise from cytosine deamination or thymine-replacing incorporation. Two enzyme activities are primarily involved in keeping DNA uracil-free: dUTPase (dUTP pyrophosphatase) activity that prevent thymine-replacing incorporation and uracil-DNA glycosylase activity that excise uracil from DNA and initiate uracil-excision repair. Both dUTPase and the most efficient uracil-DNA glycosylase (UNG) is thought to be ubiquitous in free-living organisms. In the present work, we have systematically investigated the genotype of deposited fully sequenced bacterial and Archaeal genomes. We have performed bioinformatic searches in these genomes using the already well described dUTPase and UNG gene sequences. For dUTPases, we have included the trimeric all-beta and the dimeric all-alpha families and also, the bifunctional dCTP (deoxycytidine triphosphate) deaminase-dUTPase sequences. Surprisingly, we have found that in contrast to the generally held opinion, a wide number of bacterial and Archaeal species lack all of the previously described dUTPase gene(s). The dut- genotype is present in diverse bacterial phyla indicating that loss of this (or these) gene(s) has occurred multiple times during evolution. We discuss potential survival strategies in lack of dUTPases, such as simultaneous lack or inhibition of UNG and possession of exogenous or alternate metabolic enzymes involved in uracil-DNA metabolism. The potential that genes previously not associated with dUTPase activity may still encode enzymes capable of hydrolyzing dUTP is also discussed. Our data indicate that several unicellular microorganisms may efficiently cope with a dut- genotype lacking all of the previously described dUTPase genes, and potentially leading to an unusual uracil-enrichment in their genomic DNA.

Keywords: U-DNA; UGI; Uracil-DNA-glycosylase; dUTPase; genome analysis; horizontal gene transfer; prokaryotes; uracil.

FIGURE 1

Pathways and protein factors involved in the metabolism of uracil-substituted DNA. Uracil may arise in the DNA by cytosine deamination and by dUTP incorporation. The scheme illustrates that dUTPase and UDG are responsible for keeping uracil out of DNA by dNTP pool sanitization or uracil-excision, respectively. Inhibitor proteins against UDG (UGI, SaUGI, and p56) and dUTPase (Stl) are also included on the figure, showing their point of inhibitory attack. The figure also highlights that high uracil content of the DNA can lead to strand breaks, and thus to genomic instability due to the futile cycles of base excision repair.

FIGURE 2

The distribution of bacterial/Archaeal genomes with and without dUTPase at the phylum level. Only those phyla are shown that have at least 15 genomes examined. Each node of the tree is labeled by three numbers: the first is the number of genomes with dUTPase under the node (lilac color on the pie graph segment); the second is the number of genomes without both dUTPase and UNG (blue color on the pie graph segment); the third is the number of genomes without dUTPase and with UNG (pink color on the pie graph segment).

FIGURE 3

Genomic uracil-DNA content of Staphylococcus aureus RN450, Escherichia coli (ATCC 25922), and Aeromonas hydrophila (ATCC 7966) strains. Results were obtained using the uracil-DNA quantification method as described previously (Rona et al., 2016). Significant increase (^∗) in uracil-DNA content was observed in the data for the S. aureus 450 strain as compared to the E. coli and Aeromonas hydrophila strains (P < 0.05). Calculations were based on three independent datasets, representing three different biological samples.