Domain closure and action of uracil DNA glycosylase (UDG): structures of new crystal forms containing the Escherichia coli enzyme and a comparative study of the known structures involving UDG

The structures of a new crystal form of free Escherichia coli uracil DNA glycosylase (UDG), containing four molecules in the asymmetric unit, and two forms of its complex with the proteinaceous inhibitor Ugi, containing two and four crystallographically independent complexes, have been determined. A comparison of these structures and the already known crystal structures containing UDG shows that the enzyme can be considered to be made up of two independently moving structural entities or domains. A detailed study of free and DNA-bound human enzyme strengthens this conclusion. The domains close upon binding to uracil-containing DNA, whereas they do not appear to do so upon binding to Ugi. The comparative study also shows that the mobility of the molecule involves the rigid-body movement of the domains superposed on flexibility within domains

Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island

Background: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Methodology/Principal Findings: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg 2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity

Differential effects of single-stranded DNA binding proteins (SSBs) on uracil DNA glycosylases (UDGs) from Escherichia coli and mycobacteria.

Deamination of cytosines results in accumulation of uracil residues in DNA, which unless repaired lead to GC-->AT transition mutations. Uracil DNA glyco-sylase excises uracil residues from DNA and initiates the base excision repair pathway to safeguard the genomic integrity. In this study, we have investigated the effect of single-stranded DNA binding proteins (SSBs) from Escherichia coli (Eco SSB) and Mycobacterium tuberculosis (Mtu SSB) on uracil excision from synthetic substrates by uracil DNA glycosylases (UDGs) from E. coli, Mycobacterium smegmatis and M.tuberculosis (referred to as Eco -, Msm - and Mtu UDGs respectively). Presence of SSBs with all the three UDGs resulted in decreased efficiency of uracil excision from a single-stranded 'unstructured' oligonucleo-tide, SS-U9. On the other hand, addition of Eco SSB to Eco UDG, or Mtu SSB to Mtu UDG reactions resulted in increased efficiency of uracil excision from a hairpin oligonucleotide containing dU at the second position in a tetraloop (Loop-U2). Interestingly, the efficiency of uracil excision by Msm UDG from the same substrate was decreased in the presence of either Eco- or Mtu SSBs. Furthermore, Mtu SSB also decreased uracil excision from Loop-U2 by Eco UDG. Our studies using surface plasmon resonance technique demonstrated interactions between the homologous combinations of SSBs and UDGs. Heterologous combinations either did not show detectable interaction (Eco SSB with Mtu UDG) or showed a relatively weaker interaction (Mtu SSB with Eco UDG). Taken together, our studies suggest differential interactions between the two groups (SSBs and UDGs) of the highly conserved proteins. Such studies may provide important clues to design selective inhibitors against this important class of DNA repair enzymes

Use of sequence microdivergence in mycobacterial ortholog to analyze contributions of the water-activating loop histidine of Escherichia coli uracil–DNA glycosylase in reactant binding and catalysis

Uracil–DNA glycosylase (Ung), a DNA repair enzyme, pioneers uracil excision repair pathway. Structural determinations and mutational analyses of the Ung class of proteins have greatly facilitated our understanding of the mechanism of uracil excision from DNA. More recently, a hybrid quantum-mechanical/molecular mechanical analysis revealed that while the histidine (H67 in EcoUng) of the GQDPYH motif ($\omega$ loop) in the active site pocket is important in positioning the reactants, it makes an unfavorable energetic contribution (penalty) in achieving the transition state intermediate. Mutational analysis of this histidine is unavailable from any of the Ung class of proteins. A complication in demonstrating negative role of a residue, especially when located within the active site pocket, is that the mutants with enhanced activity are rarely obtained. Interestingly, unlike the most Ung proteins, the H67 equivalent in the $\omega$ loop in mycobacterial Ung is represented by P67. Exploiting this natural diversity to maintain structural integrity of the active site, we transplanted an H67P mutation in EcoUng. Uracil inhibition assays and binding of a proteinaceous inhibitor, Ugi (a transition state substrate mimic), with the mutant (H67P) revealed that its active site pocket was not perturbed. The catalytic efficiency $(V_{max}/K_m)$ of the mutant was similar to that of the wild type Ung. However, the mutant showed increased $K_m$ and $V_{max.}$ Together with the data from a double mutation H67P/G68T, these observations provide the first biochemical evidence for the proposed diverse roles of H67 in catalysis by Ung

X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG.

Uracil-DNA glycosylase (UDG), a key highly conserved DNA repair enzyme involved in uracil excision repair, was discovered in Escherichia coli . The Bacillus subtilis bacteriophage, PBS-1 and PBS-2, which contain dUMP residues in their DNA, express a UDG inhibitor protein, Ugi which binds to UDG very tightly to form a physiologically irreversible complex. The X-ray analysis of the E. coli UDG ( Ec UDG)-Ugi complex at 3.2 A resolution, leads to the first structure elucidation of a bacterial UDG molecule. This structure is similar to the enzymes from human and viral sources. A comparison of the available structures involving UDG permits the delineation of the constant and the variable regions of the molecule. Structural comparison and mutational analysis also indicate that the mode of action of the enzyme from these sources are the same. The crystal structure shows a remarkable spatial conservation of the active site residues involved in DNA binding in spite of significant differences in the structure of the enzyme-inhibitor complex, in comparison with those from the mammalian and viral sources. Ec UDG could serve as a prototype for UDGs from pathogenic prokaryotes, and provide a framework for possible drug development against such pathogens with emphasis on features of the molecule that differ from those in the human enzyme

Crystallization and preliminary X-ray studies of the single-stranded DNA-binding protein from Mycobacterium tuberculosis

Single-stranded DNA-binding proteins play an important role in DNA replication, repair and recombination. The protein from Mycobacterium tuberculosis (MtSSB) is a tetramer with 164 amino-acid residues in each subunit. The protein readily crystallizes in space group P3<SUB>1</SUB>21 (or P3<SUB>2</SUB>21) at pH 7.4 under appropriate conditions. Under different conditions, but at the same pH, orthorhombic crystals belonging to space group I222 or I2<SUB>1</SUB>2<SUB>1</SUB>2<SUB>1</SUB> were obtained after several months. Similar orthorhombic crystals were obtained when protein samples stored for several months were used for crystallization. The orthorhombic crystals obtained in different experiments, though similar to one another, exhibited variations in unit-cell parameters, presumably on account of different extents of proteolytic cleavage of the C-terminal region. Molecular-replacement calculations using different search models did not yield the structure. As part of attempts to solve the structure using isomorphous replacement, a good mercury derivative of the trigonal crystal has been prepared

Domain closure and action of uracil DNA glycosylase (UDG): structures of new crystal forms containing the Escherichia coli enzyme and a comparative study of the known structures involving UDG

The structures of a new crystal form of free Escherichia coli uracil DNA glycosylase (UDG), containing four molecules in the asymmetric unit, and two forms of its complex with the proteinaceous inhibitor Ugi, containing two and four crystallographically independent complexes, have been determined. A comparison of these structures and the already known crystal structures containing UDG shows that the enzyme can be considered to be made up of two independently moving structural entities or domains. A detailed study of free and DNA-bound human enzyme strengthens this conclusion. The domains close upon binding to uracil-containing DNA, whereas they do not appear to do so upon binding to Ugi. The comparative study also shows that the mobility of the molecule involves the rigid-body movement of the domains superposed on flexibility within domains