Mechanistic studies on uracil DNA glycosylases from escherichia coli and mycobacteria : Interaction with uracil containing DNA, Single Stranded DNA Binding protiens(SSBs) and an Inhibitor protein(Ugi)

Abstract

Mutational and Biochemical Analyses of Uracil-DNA Glycosylase (UDG), Single-Stranded DNA Binding Proteins (SSB), and Ugi

Abstract and Synopsis Introduction Uracils arise in DNA either by spontaneous deamination of cytosine or by misincorporation of dUMP during replication. The resulting G:U mismatches are promutagenic and, if unrepaired, lead to GC AT mutations. Cells maintain genomic integrity through uracil-DNA glycosylase (UDG), a base excision repair enzyme with high turnover and strict specificity for uracil. UDG interacts with proteins such as the phage-encoded inhibitor Ugi, and cellular factors like SSB and PCNA, forming a model system to study protein-DNA and protein-protein interactions.

This study focuses on mutational analyses of E. coli UDG (EcoUDG), Mycobacterium smegmatis SSB (MsmSSB), and Ugi, to understand their structural and functional roles.

A. Mutational Analyses of EcoUDG

Crystal structures highlight conserved residues Y66 and N123 in uracil recognition.

Mutations at N123 (N123D, N123E, N123Q) revealed substrate position-dependent excision, suggesting long-range interactions.

Phosphate ethylation assays confirmed critical roles of -1, +1, +2 phosphates, and unexpectedly, the -5 phosphate.

Mutations at Y66 (Y66C, Y66S) reduced activity ~1000-fold, indicating catalytic involvement beyond base selectivity.

Further mutants (Y66F, Y66H, Y66L, Y66W) showed varied activity, with hydrophobic residues (F, L) maintaining wild-type function, while W and H reduced activity significantly.

Product inhibition studies revealed uracil and abasic DNA as inhibitors, except in Y66W, which remained uninhibited.

B. Characterization of MsmSSB

SSBs are essential for DNA metabolism, with EcoSSB’s acidic C-terminal tail mediating protein interactions.

EcoSSB enhances uracil excision by EcoUDG from hairpin substrates, while MtuSSB stimulates MtuUDG but inhibits MsmUDG.

Cloned and purified MsmSSB showed oligomerization and DNA binding similar to EcoSSB and MtuSSB.

Unlike MtuSSB, MsmSSB stimulated activity of both homologous and heterologous UDGs.

Deletion studies confirmed that the acidic tail of SSB interacts within the DNA-binding groove of UDGs.

C. Mutational Analyses of Ugi

Ugi forms stable complexes with UDGs, acting as a transition-state substrate analogue.

Mutational studies revealed:

1 helix deletions did not affect stability.

Hydrophobic pocket mutations (M24K) reduced stability, while others (E49G, D61G, N76K) had minimal effects.

Interface mutations (L23R, S21P, N35D) destabilized complexes, but compensatory mutations restored stability.

Complexes with mycobacterial UDGs were less stable (5-6 M urea) compared to EcoUDG (8 M urea), and Ugi could be displaced by EcoUDG or DNA substrate.

Findings suggest potential for Ugi-derived peptides as inhibitors of mycobacterial UDG, relevant to targeting pathogens like M. tuberculosis.

Conclusions Y66 plays a dual role in EcoUDG: base selectivity and catalysis.

MsmSSB uniquely stimulates UDG activity across species, unlike other SSBs.

Ugi mutational studies reveal structural flexibility and potential therapeutic applications.

Together, these analyses deepen understanding of UDG-mediated DNA repair and its regulation by protein partners.