ABSTRACT
Base excision repair (BER) is the primary DNA repair pathway that corrects base lesions that arise due to oxidative, alkylation, deamination, and depurinatiation/depyrimidination damage. BER facilitates the repair of damaged DNA via two general pathways - short-patch and long-patch. The shortpatch BER pathway leads to a repair tract of a single nucleotide. Alternatively, the long-patch BER pathway produces a repair tract of at least two nucleotides. The BER pathway is initiated by one of many DNA glycosylases, which recognize and catalyze the removal of damaged bases. The completion of the BER pathway is accomplished by the coordinated action of at least three additional enzymes. These downstream enzymes carry out strand incision, gap-filling and ligation. The high degree of BER conservation between E. coli and mammals has lead to advances in our understanding of mammalian BER. This review will provide a general overview of the mammalian BER pathway. (Part of a Multi-author Review).
Subject(s)
DNA Damage/physiology , DNA Glycosylases/metabolism , DNA Repair Enzymes/metabolism , DNA Repair/physiology , Models, Molecular , Alkylation , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Breaks, Single-Stranded , DNA Glycosylases/chemistry , DNA Glycosylases/genetics , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Molecular Sequence Data , Oxidation-Reduction , Protein ConformationABSTRACT
Uracil-DNA glycosylase (UDG) is a DNA-repair enzyme involved in the removal of uracil from DNA. The Atlantic cod UDG (cUDG) possesses typical cold-adaptation features, with higher catalytic efficiency and lower thermal stability than the mammalian counterparts. cUDG has been crystallized by the vapour-diffusion method using sodium citrate as the precipitant at pH 7.5. The crystals are monoclinic and belong to space group P2(1), with unit-cell parameters a = 68.58, b = 67.19, c = 68.64 A, beta = 119.85 degrees. There are two molecules in the asymmetric unit, with a corresponding V(M) value of 2.71 A(3) Da(-1) and a solvent content of 54.7%. Synchrotron diffraction data have been collected to 1.9 A resolution using cryogenic conditions (120 K).
Subject(s)
Chordata, Nonvertebrate/enzymology , DNA Glycosylases , N-Glycosyl Hydrolases/chemistry , Animals , Cold Climate , Crystallization , Crystallography, X-Ray , Protein Conformation , Uracil-DNA GlycosidaseABSTRACT
BACKGROUND: The phospholipase D (PLD) superfamily includes enzymes that are involved in phospholipid metabolism, nucleases, toxins and virus envelope proteins of unknown function. PLD hydrolyzes the terminal phosphodiester bond of phospholipids to phosphatidic acid and a hydrophilic constituent. Phosphatidic acid is a compound that is heavily involved in signal transduction. PLD also catalyses a transphosphatidylation reaction in the presence of phosphatidylcholine and a short-chained primary or secondary alcohol. RESULTS: The first crystal structure of a 54 kDa PLD has been determined to 1.9 A resolution using the multiwavelength anomalous dispersion (MAD) method on a single WO(4) ion and refined to 1.4 A resolution. PLD from the bacterial source Streptomyces sp. strain PMF consists of a single polypeptide chain that is folded into two domains. An active site is located at the interface between these domains. The presented structure supports the proposed superfamily relationship with the published structure of the 16 kDa endonuclease from Salmonella typhimurium. CONCLUSIONS: The structure of PLD provides insight into the structure and mode of action of not only bacterial, plant and mammalian PLDs, but also of a variety of enzymes as diverse as cardiolipin synthases, phosphatidylserine synthases, toxins, endonucleases, as well as poxvirus envelope proteins having a so far unknown function. The common features of these enzymes are that they can bind to a phosphodiester moiety, and that most of these enzymes are active as bi-lobed monomers or dimers.
Subject(s)
Phospholipase D/chemistry , Amino Acid Sequence , Animals , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Phospholipase D/genetics , Phospholipase D/metabolism , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Static Electricity , Streptomyces/enzymology , Streptomyces/geneticsABSTRACT
Crystals of purified phospholipase D (E.C. 3.1.4.4) from Streptomyces sp. strain PMF have been grown under two different crystallization conditions using vapour diffusion. Both conditions gave monoclinic crystals in space group P2(1). The unit-cell parameters were a = 57.28, b = 57.42, c = 68.70 A, beta = 93.17 degrees. The crystals diffract at 110 K to a resolution beyond 1.4 A using synchrotron radiation.
Subject(s)
Phospholipase D/chemistry , Streptomyces/enzymology , Crystallization , Crystallography, X-Ray , Models, Molecular , Phospholipase D/isolation & purification , Protein ConformationABSTRACT
The complex formed between anionic salmon trypsin (ST) and bovine pancreatic trypsin inhibitor (BPTI) has been crystallised, and the X-ray structure has been solved using the molecular replacement method. The crystals are hexagonal and belong to space group P6(1)22 with lattice parameters of a = b = 83.12 A and c = 222.15 A. Data have been collected to 2.1 A and the structure has been refined to a crystallographic R-factor of 20.6%. Catalysis by salmon trypsin is distinguished by a Km value 20-fold lower than that for mammalian trypsins, and a k(cat) twice as high. The present ST-BPTI complex serves as a model for the Michaelis-Menten complex, and has been compared with corresponding bovine and rat trypsin (RT) complexes. The binding of BPTI to salmon trypsin is characterised by stronger primary interactions in the active site, and a somewhat looser secondary binding.
