title: Mechanistic insights into the roles of three linked single-stranded template binding residues of MMLV reverse transcriptase in misincorporation and mispair extension fidelity of DNA synthesis.
abstract: To obtain some insights into the structure-function relationship of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT), we modeled the catalytic state ternary complexes of this protein using the corresponding RT from human immunodeficiency virus type 1 (HIV-1) and available structures of MMLV RT. We observed that three MMLV RT single-stranded template binding residues, Y64, D114, and R116, act as a linked set through mutual interactions, including hydrogen bonding and ion-pairing. The analogous residues of HIV-1 RT have a somewhat different environment and they lack this linked phenomenon. To understand the functional implication of this linked set of MMLV RT, we performed site-directed mutagenesis at these three positions. Then the mutant enzymes were examined for their biochemical properties and nucleotide selectivity. Mutagenesis of these residues (Y64A, D114A, and R116A) resulted in enzymes with slight to modest changes in polymerase activities. The processivity of DNA synthesis correlated positively with the binding affinity of the MMLV RT variants. Lower fidelity in mutants was indicated by measurements of misincorporation and mispair extension fidelity of wild type (WT) and mutant RTs, in contrast to earlier works that indicate that mutations at the analogous positions in HIV-1 RT result in relatively higher fidelity. These data together with structural analysis suggest that this structural set may therefore be a key factor responsible for the different fidelity of these two RTs.
authors.affiliation: National Key Laboratory of Respiratory Disease, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Xie
authors.forename: Pengwei
authors.initials: P
authors.lastname: Zhang
authors.forename: Chuanjiang
authors.initials: C
authors.lastname: Li
authors.forename: Qianhua
authors.initials: Q
authors.lastname: Huang
authors.forename: Rong
authors.initials: R
authors.lastname: Zhou
authors.forename: Tao
authors.initials: T
authors.lastname: Peng
mesh_terms: D054303:HIV Reverse Transcriptase / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000134:cerebrospinal fluid* / Q000378:metabolism; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
chemical_list: D050505:Mutant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase