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

Abstract

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 .

Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells

“…It is noticeable that R126C and K345E substitutions compromise only dRP lyase activity without interference with polymerase activity. A recent study suggests a role of the N-terminal region involving Gln-84 and Lys-85 residues in the dRP lyase activity of pol ι, possibly by stabilizing protein/DNA conformations required for cleavage of dRP group . We note that Arg-126 and Lys-345 residues are located not very far from Gln-84 and Lys-85 in the pol ι catalytic core structure, implying some potential dRP lyase-specific roles of these residues.…”

“…DNA substrates were prepared for polymerase and binding assays as described, using the 24-mer (5′-GCC TCG AGC CAG CCG CAG ACG CAG-3′), 36-mer (3′-CGG AGC TCG GTC GGC GTC TGC GTC XCT CCT GCG GCT-5′; X = G, 8-oxoG), and 18-FAM-mer (5′-(FAM)-AGC CAG CCG CAG ACG CAG-3′; FAM = 6-carboxyfluorescein). DNA substrates containing a 5′-dRP group were prepared for dRP lyase assays using the uracil-containing 32-mer (5′-CTG AGC AGT CGC ACA UGT AGT ATC TCT GTG AC-3′) and its complementary oligonucleotide as described …”

“…The dRP lyase assay was performed as described with minor modification . The reaction mixture contained 50 mM HEPES-KOH (pH 7.5), 20 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 100 nM DNA substrate, and 250 nM pol ι.…”

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

“…The development of KOs or gene-edited cells for Polβ has not only confirmed its involvement in nuclear BER/SSBR [366], but also in mitochondrial BER [389] and have been used to show that Polβ controls XRCC1 complex dynamics following DNA damage [366] and that Polβ is not required for HIV-1 infection [390]. While Polβ is the predominant DNA polymerase involved in BER, a role for DNA polymerase iota has been suggested [391] due to its 5 dRP lyase activity [392][393][394], its role in response to oxidative stress [395], and repair of clustered base damage [396,397]. However, a possible role for such low fidelity DNA polymerases in BER such as Pols iota, eta, or kappa, has also been disputed [398].…”

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

“…Pol ι bypasses a variety of DNA lesions with different accuracies and efficiencies [reviewed in [228]]. Like Pol β and Pol λ, Pol ι also possesses the dRP-lyase activity [236,237].…”