Format

Send to

Choose Destination
FEBS Open Bio. 2019 Feb 9;9(3):420-427. doi: 10.1002/2211-5463.12567. eCollection 2019 Mar.

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

Author information

1
Hungarian Academy of Sciences Research Centre for Natural Sciences Institute of Enzymology Budapest Hungary.
2
Department of Applied Biotechnology and Food Science Budapest University of Technology and Economics Budapest Hungary.
3
Hungarian Academy of Sciences Research Centre for Natural Sciences Institute of Organic Chemistry Budapest Hungary.
4
Department of Medical Biochemistry Semmelweis University Budapest Hungary.

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.

KEYWORDS:

S. aureus uracil‐DNA glycosylase inhibitor; SAUDG; SAUGI; base excision repair; mass spectrometry; protein complex formation

Supplemental Content

Full text links

Icon for Wiley Icon for PubMed Central
Loading ...
Support Center