Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification.

Marina Alexeeva,Arne Klungland,Svein Bjelland,Anette Rasmussen,Ingar Leiros,Xiang Ming Xu,Hilde Nilsen,Lene Alsøe,Marivi Nabong Moen,Finn Kirpekar

doi:10.3389/fimmu.2021.762032

Abstract

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.

Highlights

Hydrolytic deamination of cytosine (C) to uracil (U) in DNA is a frequent event in all including human cells
EcUng was obtained from NEB, Fermentas and Trevigen; EcNfo was obtained from Fermentas; E. coli formamidopyrimidineDNA glycosylase (EcFpg), EcNth, hOGG1, and human AP endonuclease 1 (hAPE1) were obtained from NEB. hUNGD84 mutant proteins were purified as described [41] with the following minor modifications: bacteria were grown in 0.5 or 1 l of Terrific Broth (TB) medium containing 100 mg/ml ampicillin and lysed by sonication; proteins were purified in three steps by anion and cation exchange and size exclusion chromatography using HiTrap Q (5 ml), HiTrap SP (5 ml) and HiLoad Superdex 75 16/600 columns, respectively, in that order
In this report we demonstrate that hUNG—hitherto regarded as a mono-functional DNA glycosylase—is able to incise the phosphodiester backbone of DNA at the uracil site after uracil excision (Figure 1B)

Summary

Introduction

Hydrolytic deamination of cytosine (C) to uracil (U) in DNA is a frequent event in all including human cells. Human cells contain one family 1 (uracil-DNA N-glycosidase; hUNG), one family 2 (thymine-DNA glycosylase; hTDG), and one family 3 (human single-strand-selective mono-functional UDG; hSMUG1) UDG. HUNG is removing both deaminated cytosine and misincorporated uracil, with high activity for uracil in single-stranded (ss) DNA, which is enriched in replicating cells. Family 1–3 UDGs have been classified as monofunctional DNA glycosylases only able to excise the damaged base This contrasts with the bi-functional DNA glycosylases that exhibit additional lyase activity carrying out a b- or b/delimination reaction to incise the apurinic/apyrimidinic (AP) site, such incision is believed to predominantly being accomplished by human AP endonuclease 1 (hAPE1) [17,18,19] in vivo, which is able to process an AP ribonucleoside embedded in DNA [20]. The BER pathway is completed by the sequential action of DNA polymerase b [22], which removes the 5 ́-dRP by its lyase function if hAPE1 incised the AP site, and DNA ligase [1, 2, 5]

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Results

Conclusion