Mass spectrometry-based analysis of macromolecular complexes of Staphylococcus aureus uracil-DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations.

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil-DNA glycosylase. Interestingly, both uracil-DNA glycosylase (Staphylococcus aureus uracil-DNA glycosylase; SAUDG) and its inhibitor (S. aureus uracil-DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil-DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these Kd values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil-DNA glycosylase inhibitor proteins.

Structural model of human dUTPase in complex with a novel proteinaceous inhibitor.

Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein StlSaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition. Small-angle X-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data allowed us to obtain 3D structural models comprising a trimeric dUTPase complexed with separate Stl monomers. These models thus reveal that upon dUTPase-Stl complex formation the functional homodimer of Stl repressor dissociates, which abolishes the DNA binding ability of the protein. Active site forming dUTPase segments were directly identified to be involved in the dUTPase-Stl interaction by HDX-MS, explaining the loss of dUTPase activity upon complexation. Our results provide key novel structural insights that pave the way for further applications of the first potent proteinaceous inhibitor of human dUTPase.

Life without dUTPase.

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.

In Vitro Analysis of Predicted DNA-Binding Sites for the Stl Repressor of the Staphylococcus aureus SaPIBov1 Pathogenicity Island.

The regulation model of the Staphylococcus aureus pathogenicity island SaPIbov1 transfer was recently reported. The repressor protein Stl obstructs the expression of SaPI proteins Str and Xis, latter which is responsible for mobilization initiation. Upon Φ11 phage infection of S. aureus. phage dUTPase activates the SaPI transfer via Stl-dUTPase complex formation. Our aim was to predict the binding sites for the Stl repressor within the S. aureus pathogenicity island DNA sequence. We found that Stl was capable to bind to three 23-mer oligonucleotides, two of those constituting sequence segments in the stl-str, while the other corresponding to sequence segment within the str-xis intergenic region. Within these oligonucleotides, mutational analysis revealed that the predicted binding site for the Stl protein exists as a palindromic segment in both intergenic locations. The palindromes are built as 6-mer repeat sequences involved in Stl binding. The 6-mer repeats are separated by a 5 oligonucleotides long, nonspecific sequence. Future examination of the interaction between Stl and its binding sites in vivo will provide a molecular explanation for the mechanisms of gene repression and gene activation exerted simultaneously by the Stl protein in regulating transfer of the SaPIbov1 pathogenicity island in S. aureus.

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control.

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

Structure and enzymatic mechanism of a moonlighting dUTPase.

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Šresolution three-dimensional structure of a ϕ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small ß-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of ϕ11 phage dUTPase.