Papers by Yaroslava Polosina
DNA polymerase (pol ) is the major DNA polymerase involved in the base excision repair (BER) path... more DNA polymerase (pol ) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol . Pol null (−/−) mouse embryos are not viable and pol null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol protein and mRNA to undetectable levels. Pol knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol null cells. Generation of pol knock down cells makes it possible to combine the pol null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol and its interactions with other proteins in mammalian cells.
Journal of Biological Chemistry, 2005
DNA polymerase (pol) is a member of the X family of DNA polymerases that has been implicated in b... more DNA polymerase (pol) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.
Journal of Bacteriology, 2009
The Journal of Biochemistry, Jul 1, 2009
In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are c... more In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch (VSP) repair pathway. DNA Polymerase I removes and resynthesizes the mismatched T starting from a 5'-nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA Pol I releases Vsr from the nick by reversing that conformational change.
Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis, Dec 1, 2010
In model DNA, A pairs with T, and C with G. However, in vivo, the complementarity of the DNA stra... more In model DNA, A pairs with T, and C with G. However, in vivo, the complementarity of the DNA strands may be disrupted by errors in DNA replication, biochemical modification of bases and recombination. In prokaryotic organisms, mispaired bases are recognized by MutS homologs which, together with MutL homologs, initiate mismatch repair. These same proteins also participate in base excision repair and nucleotide excision repair. In eukaryotes they regulate not just DNA repair but also meiotic recombination, cell-cycle delay and/or apoptosis in response to DNA damage, and hypermutation in immunoglobulin genes. Significantly, the same DNA mismatches that trigger repair in some circumstances trigger non-repair pathways in others. In this review, we argue that mismatch recognition by the MutS proteins is linked to these disparate biological outcomes through regulated interaction of MutL proteins with a wide variety of effector proteins.
Encyclopedia of Life Sciences, 2001
Mutation Research/Reviews in Mutation Research, 2010
In model DNA, A pairs with T, and C with G. However, in vivo, the complementarity of the DNA stra... more In model DNA, A pairs with T, and C with G. However, in vivo, the complementarity of the DNA strands may be disrupted by errors in DNA replication, biochemical modification of bases and recombination. In prokaryotic organisms, mispaired bases are recognized by MutS homologs which, together with MutL homologs, initiate mismatch repair. These same proteins also participate in base excision repair and nucleotide excision repair. In eukaryotes they regulate not just DNA repair but also meiotic recombination, cell-cycle delay and/or apoptosis in response to DNA damage, and hypermutation in immunoglobulin genes. Significantly, the same DNA mismatches that trigger repair in some circumstances trigger non-repair pathways in others. In this review, we argue that mismatch recognition by the MutS proteins is linked to these disparate biological outcomes through regulated interaction of MutL proteins with a wide variety of effector proteins.
Journal of Biochemistry, 2009
In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are c... more In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch (VSP) repair pathway. DNA Polymerase I removes and resynthesizes the mismatched T starting from a 5'-nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA Pol I releases Vsr from the nick by reversing that conformational change.
Journal of Bacteriology, 2009
The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The ... more The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.
Extremophiles, 1998
An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified a... more An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange γ-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23 000) and of the oligomer determined by sedimentation equilibrium experiments (125 000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [γ-32 P]ATP, and Mg 2ϩ was required for the incorporation of phosphate. The kinase preserved the ability to transfer γ-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.
Extremophiles, 2002
Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a... more Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa. Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.
DNA Repair, 2004
DNA polymerase  (pol ) is the major DNA polymerase involved in the base excision repair (BER) p... more DNA polymerase  (pol ) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol . Pol  null (−/−) mouse embryos are not viable and pol  null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol  protein and mRNA to undetectable levels. Pol  knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol  null cells. Generation of pol  knock down cells makes it possible to combine the pol  null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol  and its interactions with other proteins in mammalian cells.
DNA Repair, 2009
a b s t r a c t 5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The re... more a b s t r a c t 5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS-MutL complex recruits the Uvr proteins to complete repair.
BioEssays, 2000
Base pair mismatches in DNA arise from errors in DNA replication, recombination, and biochemical ... more Base pair mismatches in DNA arise from errors in DNA replication, recombination, and biochemical modification of bases. Mismatches are inherently transient. They are resolved passively by DNA replication, or actively by enzymatic removal and resynthesis of one of the bases. The first step in removal is recognition of strand discontinuity by one of the MutS proteins. Mismatches arising from errors in DNA replication are repaired in favor of the base on the template strand, but other mismatches trigger base excision or nucleotide excision repair (NER), or non-repair pathways such as hypermutation, cell cycle arrest, or apoptosis. We argue that MutL homologues play a key role in determining biologic outcome by recruiting and/or activating effector proteins in response to lesion recognition by MutS. We suggest that the process is regulated by conformational changes in MutL caused by cycles of ATP binding and hydrolysis, and by physiologic changes which influence effector availability.
Journal of Biological Chemistry, 2005
DNA polymerase (pol ) is a member of the X family of DNA polymerases that has been implicated in ... more DNA polymerase (pol ) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.
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Papers by Yaroslava Polosina