Deoxyinosine repair in nuclear extracts of human cells

Lee et al. Cell Biosci (2015) 5:52 DOI 10.1186/s13578-015-0044-8 Open Access RESEARCH Deoxyinosine repair in nuclear extracts of human cells Chia‑Chia Lee1, Ya‑Chien Yang1,2, Steven D. Goodman3, Shi Chen1, Teng‑Yung Huang1, Wern‑Cherng Cheng2, Liang‑In Lin1,2 and Woei‑horng Fang1,2* Abstract Background: Deamination of adenine can occur spontaneously under physiological conditions generating the highly mutagenic lesion, hypoxanthine. This process is enhanced by ROS from exposure of DNA to ionizing radia‑ tion, UV light, nitrous acid, or heat. Hypoxanthine in DNA can pair with cytosine which results in A:T to G:C transition mutations after DNA replication. In Escherichia coli, deoxyinosine (hypoxanthine deoxyribonucleotide, dI) is removed through an alternative excision repair pathway initiated by endonuclease V. However, the correction of dI in mamma‑ lian cells appears more complex and was not fully understood. Results: All four possible dI-containing heteroduplex DNAs, including A-I, C-I, G-I, and T-I were introduced to repair reactions containing extracts from human cells. The repair reaction requires magnesium, dNTPs, and ATP as cofac‑ tors. We found G-I was the best substrate followed by T-I, A-I and C-I, respectively. Moreover, judging from the repair requirements and sensitivity to specific polymerase inhibitors, there were overlapping repair activities in processing of dI in DNA. Indeed, a hereditable non-polyposis colorectal cancer cell line (HCT116) demonstrated lower dI repair activity that was partially attributed to lack of mismatch repair. Conclusions: A plasmid-based convenient and non-radioisotopic method was created to study dI repair in human cells. Mutagenic dI lesions processed in vitro can be scored by restriction enzyme cleavage to evaluate the repair. The repair assay described in this study provides a good platform for further investigation of human repair pathways involved in dI processing and their biological significance in mutation prevention. Keywords: Deoxyinosine repair, Mismatch repair, Human cell extracts, In vitro assay, DNA repair deficiency Background Deoxyinosine (hypoxanthine deoxyribonucleotide, dI) in DNA can arise from spontaneous deamination of deoxyadenosine residue, and is also induced by ROS produced from normal aerobic respiration. In addition, exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat can promote the formation of dI [1, 2]. Alternatively, dI can be introduced by misincorporation of dITP in the nucleotide pool during replication [3, 4]. Deoxyinosine derived from deamination of deoxyadenosine in DNA is potentially mutagenic since it prefers to pair with dCTP *Correspondence: 1 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, #7, Chung‑Shan South Road, Taipei 10002, Taiwan, ROC Full list of author information is available at the end of the article during replication, yielding A:T to G:C transition mutations at sites of adenine deamination [5]. In mammalian cells, base excision repair (BER) was thought to be the major pathway for dI repair. The excision of base damage is initiated by a specific DNA glycosylase: Hypoxanthine is bound and excised efficiently by human N-methylpurine-DNA glycosylase (MPG, also known as AAG, ANPG, APNG, or MDG) [6]. From radionucleotide incorporation fine mapping, the resulting apurinic/apyrimidinic (AP) sites are further processed by both the short patch pathway (1-nucleotide gap filling) with DNA polymerase (Pol) β and the long patch pathway (2-6 nucleotide resynthesis) with Pol δ and PCNA [7]. In Escherichia coli, early studies indicated that the DNA glycosylase encoded by alkA gene could recognize and release hypoxanthine residues from DNA [1]. However, © 2015 Lee et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Cell Biosci (2015) 5:52 subsequent in vivo and in vitro studies showed that DNA glycosylase initiated BER is not the major pathway to process dI in E. coli [8, 9]. The repair pathway initiated by endonuclease V (EndoV, encoded by nfi gene [10]) was shown to be the major pathway for dI processing both in vivo and in vitro [8, 9, 11]. A mutagenesis assay also showed that under HNO2 treatment, which will promote hypoxanthine formation, that a nfi mutant demonstrated over a 200-fold increase in mutation frequency, while the alkA mutant did not significantly increase the mutation frequency under the same experimental conditions [12]. Endonuclease V (EndoV) in E. coli is active upon DNA exposed to UV light, OsO4, acids, or X-rays [10]. This enzyme was later characterized as 3′-deoxyinosine endonuclease that incises the DNA at the second phosphodiester bond 3′ to the dI lesion, leaving 3′-OH and 5′-P termini [13]. Nfi homologues from Thermotoga maritima possess 3′-exonuclease activity that might be used for removal of damaged bases [14], but similar exonuclease activities were not found in EndoV from E. coli and mammalian cells. Therefore, additional enzymes are required to excise the dI lesions in the EndoV-mediated repair process. In our previous study, we found DNA pol I played dual roles in both repair synthesis and using its 3′-5′ proofreading exonuclease to remove EndoV incised dI lesion [9, 11]. A mammalian homologue of E. coli nfi gene was identified and characterized [15]. The mouse EndoV seems to be active only on dI, while bacterial EndoV exhibits broad substrate spectrum. Furthermore, expression of mouse EndoV in an alkA, and nfi double mutant E. coli strain significantly suppresses the spontaneous mutagenesis frequency, which suggested that this eukaryotic EndoV initiates an alternative excision repair pathway for dI correction [15]. A biochemical analysis of purified human EndoV showed it favored dI-containing DNA but with only a minor preference on deoxyxanthosine-containing DNA [16]. Expression of hEndoV in E. coli cells deficient in nfi, mug and ung genes caused 3-fold reduction in mutation frequency [16]. However, recent reports demonstrated efficient cleavage of inosine-containing RNA by human EndoV [17] suggesting that hEndoV may involve in RNA editing [18]. Therefore, the full involvement of hEndoV in dI repair in human cells is still unknown. The major function of mismatch repair (MMR) is its role in correction of nucleotide base misincorporation during replication [19–21], which requires that repair be directed to a newly synthe (...truncated)