Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota

Makarova, Аlena V.; Ignatov, Artem; Miropolskaya, Nataliya; Kulbachinskiy, Andrey

doi:10.1016/j.dnarep.2014.07.006

Cited by 14 publications

(11 citation statements)

References 55 publications

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“…Proteins were eluted and stored at −80 °C in elution buffer containing 30 mM HEPES pH 8.0, 8% glycerol, 100 mM KCl, 5 mM K 2 HPO 4 /KH 2 PO 4 pH 8.0, 1 mM DTT, 30 mM glutathione. Full-length wild-type Pol ι, full-length Pol ι variants with amino acid substitutions Y39A, Q59A, K60G and Y61A as well as truncated Pol ι encoding for the first 420 amino acids were obtained earlier 12 , 20 . Full-length Pol ι variant with amino acid substitution K72A was obtained in this work using site-directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

“…To obtain DNA substrate for DNA polymerase reactions 16-mer primer was 5′-labeled with [ γ−32 P]-ATP using T4 polynucleotide kinase and annealed to unlabeled 30-mer template oligonucleotide 12 .…”

Section: Methodsmentioning

confidence: 99%

“…The high error rate of Pol ι is a result of the special organization of the active site 1 , 2 which is adapted to bypass a variety of DNA lesions 3 , including bulky carcinogenic lesions 4 – 9 and interstrand DNA cross-links 10 . The DNA polymerase activity of Pol ι is stimulated by Mn 2+ ions 11 , 12 . In addition to the DNA polymerase activity, human Pol ι has an intrinsic 5′-deoxyribose-5-phosphate lyase activity (dRP-lyase activity) 13 , 14 .…”

Section: Introductionmentioning

confidence: 99%

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

Miropolskaya

Petushkov

Kulbachinskiy

et al. 2017

Sci Rep

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Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Yand 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 involved in the dRP-lyase activity of human Pol ι. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ι 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 ι, 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 ι, with possible involvement of residues from the finger domain in the dRP group cleavage.Human DNA polymerase iota (Pol ι) belongs to the Y-family of translesion DNA polymerases and demonstrates very low accuracy of DNA synthesis. The high error rate of Pol ι is a result of the special organization of the active site 1, 2 which is adapted to bypass a variety of DNA lesions 3 , including bulky carcinogenic lesions 4-9 and interstrand DNA cross-links 10 . The DNA polymerase activity of Pol ι is stimulated by Mn 2+ ions 11,12 . In addition to the DNA polymerase activity, human Pol ι has an intrinsic 5′-deoxyribose-5-phosphate lyase activity (dRP-lyase activity) 13,14 . Pol ι carries out efficient DNA synthesis on gapped DNA substrates and in reactions reconstituted with uracil-DNA glycosylase, AP-endonuclease and ligase can repair DNA 13,15 . Moreover, Pol ι is able to complement in vitro the short-patch base excision repair (BER) deficiency of Pol β null cell extracts 16 .

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Miropolskaya

Petushkov

Kulbachinskiy

et al. 2017

Sci Rep

Self Cite

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“…The short C1 -C1 distance between templating and incoming nucleotides (< 9 Å) interferes with the formation of canonical WC base pairs (required~10 Å C1'-C1' distance) [238][239][240]. When replicating opposite template purines, Pol ι uses HG base pairing to fit the nascent base pair in the active site [238,239,241,242]. Purines are fixed in the syn-conformation even if WC pairing is energetically more favorable (e.g., WC (anti)G:(anti)dCMP pair forms three H-bonds while HG (syn)G:(anti)dCMP pair forms two H-bonds and requires protonation of the incoming dCTP) [205,239].…”

Section: Dna Polymerase ιmentioning

confidence: 99%

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

et al. 2019

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7,8-Dihydro-8-oxoguanine (oxoG) is the most abundant oxidative DNA lesion with dual coding properties. It forms both Watson–Crick (anti)oxoG:(anti)C and Hoogsteen (syn)oxoG:(anti)A base pairs without a significant distortion of a B-DNA helix. DNA polymerases bypass oxoG but the accuracy of nucleotide incorporation opposite the lesion varies depending on the polymerase-specific interactions with the templating oxoG and incoming nucleotides. High-fidelity replicative DNA polymerases read oxoG as a cognate base for A while treating oxoG:C as a mismatch. The mutagenic effects of oxoG in the cell are alleviated by specific systems for DNA repair and nucleotide pool sanitization, preventing mutagenesis from both direct DNA oxidation and oxodGMP incorporation. DNA translesion synthesis could provide an additional protective mechanism against oxoG mutagenesis in cells. Several human DNA polymerases of the X- and Y-families efficiently and accurately incorporate nucleotides opposite oxoG. In this review, we address the mutagenic potential of oxoG in cells and discuss the structural basis for oxoG bypass by different DNA polymerases and the mechanisms of the recognition of oxoG by DNA glycosylases and dNTP hydrolases.

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“…However, Mn 2+ ions decrease the fidelity of nucleotide incorporation by PrimPol [ 31 , 32 , 33 ]. Previous studies demonstrated that Mn 2+ ions alter catalytic properties of many translesion DNA polymerases and, in particular, stimulate catalysis and DNA damage bypass by human Pol ι [ 37 , 38 ], Pol λ [ 39 ] and Pol μ [ 40 ]. Mn 2+ -dependent DNA synthesis may therefore play a role in the regulation of TLS in vivo.…”

Section: Activities and Fidelity Of Primpolmentioning

confidence: 99%

DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol

Boldinova

Wanrooij

Shilkin

et al. 2017

IJMS

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PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.

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Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota

Cited by 14 publications

References 55 publications

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

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

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol

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