@article {65306, title = {A polar filter in DNA polymerases prevents ribonucleotide incorporation.}, journal = {Nucleic Acids Res}, year = {2019}, month = {2019 Sep 23}, abstract = {

The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA polymerases (dPols) possess a steric filter. The dPol named MsDpo4 (Mycobacterium smegmatis) naturally lacks this steric filter and hence is capable of rNTP addition. The introduction of the steric filter in MsDpo4 did not result in complete abrogation of the ability of this enzyme to incorporate ribonucleotides. In comparison, DNA polymerase IV (PolIV) from Escherichia coli exhibited stringent selection for deoxyribonucleotides. A comparison of MsDpo4 and PolIV led to the discovery of an additional polar filter responsible for sugar selectivity. Thr43 represents the filter in PolIV and this residue forms interactions with the incoming nucleotide to draw it closer to the enzyme surface. As a result, the 2{\textquoteright}-OH in rNTPs will clash with the enzyme surface, and therefore ribonucleotides cannot be accommodated in the active site in a conformation compatible with productive catalysis. The substitution of the equivalent residue in MsDpo4-Cys47, with Thr led to a drastic reduction in the ability of the mycobacterial enzyme to incorporate rNTPs. Overall, our studies evince that the polar filter serves to prevent ribonucleotide incorporation by dPols.

}, issn = {1362-4962}, doi = {10.1093/nar/gkz792}, author = {Johnson, Mary K and Kottur, Jithesh and Nair, Deepak T} } @article {3555, title = {The N(2)-Furfuryl-deoxyguanosine Adduct Does Not Alter the Structure of B-DNA.}, journal = {J Org Chem}, volume = {81}, year = {2016}, month = {2016 Jan 15}, pages = {502-11}, abstract = {

N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by \~{}1.6 {\textdegree}C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct.

}, issn = {1520-6904}, doi = {10.1021/acs.joc.5b02341}, author = {Ghodke, Pratibha P and Gore, Kiran R and Harikrishna, S and Samanta, Biswajit and Kottur, Jithesh and Nair, Deepak T and Pradeepkumar, P I} } @article {3572, title = {Reactive Oxygen Species Play an Important Role in the Bactericidal Activity of Quinolone Antibiotics.}, journal = {Angew Chem Int Ed Engl}, volume = {55}, year = {2016}, month = {2016 Feb}, pages = {2397-400}, abstract = {

Recent studies posit that reactive oxygen species (ROS) contribute to the cell lethality of bactericidal antibiotics. However, this conjecture has been challenged and remains controversial. To resolve this controversy, we adopted a strategy that involves DNA polymerase IV (PolIV). The nucleotide pool of the cell gets oxidized by ROS and PolIV incorporates the damaged nucleotides (especially 8oxodGTP) into the genome, which results in death of the bacteria. By using a combination of structural and biochemical tools coupled with growth assays, it was shown that selective perturbation of the 8oxodGTP incorporation activity of PolIV results in considerable enhancement of the survival of bacteria in the presence of the norfloxacin antibiotic. Our studies therefore indicate that ROS induced in bacteria by the presence of antibiotics in the environment contribute significantly to cell lethality.

}, issn = {1521-3773}, doi = {10.1002/anie.201509340}, author = {Kottur, Jithesh and Nair, Deepak T} } @article {897, title = {Fast native-SAD phasing for routine macromolecular structure determination.}, journal = {Nat Methods}, volume = {12}, year = {2015}, month = {2015 Feb}, pages = {131-3}, abstract = {

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

}, keywords = {Animals, DNA-Binding Proteins, Humans, Membrane Proteins, Models, Molecular, Multiprotein Complexes, Protein Conformation, Software, Synchrotrons, X-Ray Diffraction}, issn = {1548-7105}, doi = {10.1038/nmeth.3211}, author = {Weinert, Tobias and Olieric, Vincent and Waltersperger, Sandro and Panepucci, Ezequiel and Chen, Lirong and Zhang, Hua and Zhou, Dayong and Rose, John and Ebihara, Akio and Kuramitsu, Seiki and Li, Dianfan and Howe, Nicole and Schnapp, Gisela and Pautsch, Alexander and Bargsten, Katja and Prota, Andrea E and Surana, Parag and Kottur, Jithesh and Nair, Deepak T and Basilico, Federica and Cecatiello, Valentina and Pasqualato, Sebastiano and Boland, Andreas and Weichenrieder, Oliver and Wang, Bi-Cheng and Steinmetz, Michel O and Caffrey, Martin and Wang, Meitian} } @article {856, title = {Unique structural features in DNA polymerase IV enable efficient bypass of the N2 adduct induced by the nitrofurazone antibiotic.}, journal = {Structure}, volume = {23}, year = {2015}, month = {2015 Jan 6}, pages = {56-67}, abstract = {

The reduction in the efficacy of therapeutic antibiotics represents a global problem of increasing intensity and concern. Nitrofuran antibiotics act primarily through the formation of covalent adducts at the N(2) atom of the deoxyguanosine nucleotide in genomic DNA. These adducts inhibit replicative DNA polymerases (dPols), leading to the death of the prokaryote. N(2)-furfuryl-deoxyguanosine (fdG) represents a stable structural analog of the nitrofuran-induced adducts. Unlike other known dPols, DNA polymerase IV (PolIV) from E.\ coli can bypass the fdG adduct accurately with high catalytic efficiency. This property of PolIV is central to its role in reducing the sensitivity of E.\ coli toward nitrofuran antibiotics such as nitrofurazone (NFZ). We present the mechanism used by PolIV to bypass NFZ-induced adducts and thus improve viability of E.\ coli in the presence of NFZ. Our results can be used to develop specific inhibitors of PolIV that may potentiate the activity of nitrofuran antibiotics.

}, keywords = {Anti-Bacterial Agents, Crystallography, X-Ray, DNA Adducts, DNA Polymerase beta, Drug Resistance, Bacterial, Escherichia coli, Escherichia coli Proteins, Models, Molecular, Nitrofurazone, Nitrogen Compounds, Protein Structure, Tertiary, Reactive Nitrogen Species}, issn = {1878-4186}, doi = {10.1016/j.str.2014.10.019}, author = {Kottur, Jithesh and Sharma, Amit and Gore, Kiran R and Narayanan, Naveen and Samanta, Biswajit and Pradeepkumar, Pushpangadan I and Nair, Deepak T} }