Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

Abstract

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 K d 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.

Viruses with U-DNA: New Avenues for Biotechnology

“…The first one in 2006 was the protein p56 of Bacillus phage Φ29 [ 50 ]. More recently, the UNG inhibitor of Staphylococcus aureus was discovered starting from bioinformatic analysis of protein structure data, and based on its origin, was termed as Staphylococcus aureus UGI, abbreviated as SaUGI [ 45 , 51 ].…”

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity

“…This protein was heat activated for 15 min at 95 °C before the ITC measurement. Furthermore, we used our previously cloned N -terminally His-tagged Staphylococcus aureus uracil glycosylase inhibitor (SaUGI) [21] and Petroselinum crispum phenylalanine ammonia-lyase (PcPAL) [22] proteins. The rabbit skeletal myosin subfragment-S1 was a kind gift of Máté Gyimesi, Eötvös University, Budapest, Hungary.…”