title: A water-mediated and substrate-assisted catalytic mechanism for Sulfolobus solfataricus DNA polymerase IV.
abstract: DNA polymerases are enzymes responsible for the synthesis of DNA from nucleotides. Understanding their molecular fundamentals is a prerequisite for elucidating their aberrant activities in diseases such as cancer. Here we have carried out ab initio quantum mechanical/molecular mechanical (QM/MM) studies on the nucleotidyl-transfer reaction catalyzed by the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus, with template guanine and Watson-Crick paired dCTP as the nascent base pair. The results suggested a novel water-mediated and substrate-assisted (WMSA) mechanism: the initial proton transfer to the alpha-phosphate of the substrate via a bridging crystal water molecule is the rate-limiting step, the nucleotidyl-transfer step is associative with a metastable pentacovalent phosphorane intermediate, and the pyrophosphate leaving is facilitated by a highly coordinated proton relay mechanism through mediation of water which neutralizes the evolving negative charge. The conserved carboxylates, which retain their liganding to the two Mg2+ ions during the reaction process, are found to be essential in stabilizing transition states. This WMSA mechanism takes specific advantage of the unique structural features of this low-fidelity lesion-bypass Y-family polymerase, which has a more spacious and solvent-exposed active site than replicative and repair polymerases.
authors: Biology and Chemistry Departments, New York University, New York, NY 10003, USA.
authors: Lihua
authors: L
authors: Wang
authors: Xinyun
authors: X
authors: Yu
authors: Po
authors: P
authors: Hu
authors: Suse
authors: S
authors: Broyde
authors: Yingkai
authors: Y
authors: Zhang
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D014867:Water / Q000737:chemistry*
chemical_list: D014867:Water; D019951:DNA Polymerase beta