ABSTRACT
Many chemotherapy drugs block tumor cell division by damaging DNA. DNA polymerases eta (Pol η), iota (Pol ι), kappa (Pol κ), REV1 of the Y-family and zeta (Pol ζ) of the B-family efficiently incorporate nucleotides opposite a number of DNA lesions during translesion DNA synthesis. Primase-polymerase PrimPol and the Pol α-primase complex reinitiate DNA synthesis downstream of the damaged sites using their DNA primase activity. These enzymes can decrease the efficacy of chemotherapy drugs, contribute to the survival of tumor cells and to the progression of malignant diseases. DNA polymerases are promising targets for increasing the effectiveness of chemotherapy, and mutations and polymorphisms in some DNA polymerases can serve as additional prognostic markers in a number of oncological disorders.
Subject(s)
Antineoplastic Agents/pharmacology , DNA-Directed DNA Polymerase/metabolism , DNA/metabolism , Drug Resistance, Neoplasm , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein Biosynthesis/drug effects , Animals , Antineoplastic Agents/therapeutic use , DNA Damage/drug effects , DNA Repair/drug effects , DNA Replication/drug effects , DNA-Directed DNA Polymerase/genetics , Humans , Neoplasms/drug therapy , Nucleic Acid Synthesis Inhibitors/therapeutic use , Polymorphism, GeneticABSTRACT
Tens of thousands of DNA lesions are formed in mammalian cells each day. DNA translesion synthesis is the main mechanism of cell defense against unrepaired DNA lesions. DNA polymerases iota (Pol ι), eta (Pol η), kappa (Pol κ), and zeta (Pol ζ) have active sites that are less stringent toward the DNA template structure and efficiently incorporate nucleotides opposite DNA lesions. However, these polymerases display low accuracy of DNA synthesis and can introduce mutations in genomic DNA. Impaired functioning of these enzymes can lead to an increased risk of cancer.
Subject(s)
DNA Damage , DNA Repair , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Neoplasms/pathology , Animals , Humans , Neoplasms/genetics , Neoplasms/metabolism , DNA Polymerase iotaABSTRACT
The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.
Subject(s)
Bacteriophages/enzymology , N-Acetylmuramoyl-L-alanine Amidase/genetics , Pseudomonas aeruginosa/virology , Amino Acid Sequence , Bacteriophages/ultrastructure , Enzyme Stability , Genome , Humans , Molecular Sequence Data , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Pseudomonas aeruginosa/genetics , Sequence Analysis, DNAABSTRACT
The key stage of the infection of the Escherichia coli cell with bacteriophage T4, the binding to the surface of the host cell, is determined by the specificity of the long tail fiber proteins of the phage, in particular, gp37. The assembly and oligomerization of this protein under natural conditions requires the participation of at least two additional protein factors, gp57A and gp38, which strongly hinders the production of the recombinant form of gp37. To overcome this problem, a modern protein engineering strategy was used, which involves the construction of a chimeric protein containing a carrier protein that drives the correct folding of the target protein. For this purpose, the trimeric beta-helical domain of another protein of phage T4, gp5, was used. It was shown that this domain, represented as a rigid trimeric polypeptide prism, has properties favorable for use as a protein carrier. A fragment of protein gp37 containing five pentapeptides repeats, Gly-X-His-X-His, which determine the binding to the receptors on the bacterial cell surface, was fused in a continuous reading frame to the C-terminus of the domain of gp5. The resulting chimeric protein forms a trimer that has the native conformation of gp37 and exhibits biological activity.
Subject(s)
Bacteriophage T4/genetics , Escherichia coli/metabolism , Recombinant Fusion Proteins/biosynthesis , Viral Proteins/genetics , Bacteriophage T4/physiology , Escherichia coli/genetics , Escherichia coli/virology , Models, Molecular , Protein Engineering , Protein Folding , Protein Multimerization , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Viral Tail Proteins/biosynthesis , Viral Tail Proteins/genetics , Viral Tail Proteins/isolation & purificationABSTRACT
Attempts to crystallize the noncovalent complex of recombinant human insulin with polysialic acid were carried out under normal and microgravity conditions. Both crystal types belonged to the same space group, I2(1)3, with unit-cell parameters a = b = c = 77.365 A, alpha = beta = gamma = 90.00 degrees. The reported space group and unit-cell parameters are almost identical to those of cubic insulin reported in the PDB. The results of X-ray studies confirmed that the crystals obtained were cubic insulin crystals and that they contained no polysialic acid or its fragments. Electron-density maps were calculated using X-ray diffraction sets from earth-grown and microgravity-grown crystals and the three-dimensional structure of the insulin molecule was determined and refined. The conformation and secondary-structural elements of the insulin molecule in different crystal forms were compared.
