Dataset Information

Identification of amino acid residues involved in the dRP-lyase activity of human Pol ?.

ABSTRACT: Besides X-family DNA polymerases (first of all, Pol ?) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ?, Rev1, Pol ? and Pol ?). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly involved in the dRP-lyase activity of human Pol ?. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ? while residues in the HhH motif of the thumb domain are dispensable for this activity. While both K60G and K207A substitutions decrease Schiff-base intermediate formation during dRP group cleavage, the latter substitution also strongly affects the DNA polymerase activity of Pol ?, suggesting that it may impair DNA binding. These data are consistent with an important role of the N-terminal region in the dRP-lyase activity of Pol ?, with possible involvement of residues from the finger domain in the dRP group cleavage.

SUBMITTER: Miropolskaya N

PROVIDER: S-EPMC5579206 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Identification of amino acid residues involved in the dRP-lyase activity of human Pol ι.

Miropolskaya Nataliya N Petushkov Ivan I Kulbachinskiy Andrey A Makarova Alena V AV

Scientific reports 20170831 1

Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ι, Rev1, Pol γ and Pol θ). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly invol ...[more]

PMID: 28860552

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Project description:GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-?-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent ?-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.

Dataset Information

Identification of amino acid residues involved in the dRP-lyase activity of human Pol ?.

Publications

Identification of amino acid residues involved in the dRP-lyase activity of human Pol ι.

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