Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in Mycobacterium tuberculosis and Is Important for Protection against Oxidative Damage Rupangi Verma Puri, Nisha Singh, Rakesh K. Gupta ¤ , Anil K. Tyagi* Department of Biochemistry, University of Delhi South Campus, New Delhi, India Abstract During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER) pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP) endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End) and Exonuclease III (XthA) that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 39R59 exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg 2+ and Ca 2+ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 39R59 exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress. Citation: Puri RV, Singh N, Gupta RK, Tyagi AK (2013) Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in Mycobacterium tuberculosis and Is Important for Protection against Oxidative Damage. PLoS ONE 8(8): e71535. doi:10.1371/journal.pone.0071535 Editor: Tanya Parish, Infectious Disease Research Institute, United States of America Received April 7, 2013; Accepted June 29, 2013; Published August 1, 2013 Copyright: ß 2013 Puri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a research grant from the Department of Biotechnology, Ministry of Science and Technology, Government of India. The Council of Scientific and Industrial Research, Government of India, is acknowledged for Junior/Senior research fellowships to RVP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: AT is a member of the PLOS ONE Editorial Board. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: aniltyagi@south.du.ac.in ¤ Current address: Department of Microbiology, Ram Lal Anand College, University of Delhi South Campus, New Delhi, India Introduction Cellular DNA is subjected to continuous assaults by a wide variety of intimidating endogenous and exogenous agents. Amongst these, the predominant damage is caused by reactive oxygen species (ROS) that are formed as by-products of oxidative metabolism in organisms with aerobic respiration. Exogenous agents such as chemical carcinogens and ionizing radiation also generate ROS. These oxygen radicals mostly produce non-bulky DNA lesions that are substrates for the base excision repair (BER) pathway [1,2]. This pathway is highly conserved from bacteria to humans [3]. Apurinic and apyrimidinic (AP) sites (also known as abasic sites), are the most common DNA lesions that arise during the spontaneous loss of normal or damaged bases or by the action of DNA glycosylases that release modified or mismatched bases from DNA [4,5,6]. The AP sites threaten genetic stability because they block replication and are mutagenic [7,8]. Thus, the removal of AP sites in DNA by a Class II AP endonuclease is a crucial step in BER. AP endonuclease enzymes initiate the repair of abasic sites in DNA by cleavage of the DNA backbone immediately 59 of an AP residue. A single-nucleotide gap is then created in the DNA by the action of 59 deoxyribose phosphodiesterase. The base excision repair is completed by resynthesis of the DNA by DNA polymerase followed by the action of DNA ligase [9]. The Class II AP endonucleases have been classified into two families, the exonuclease III (ExoIII) and endonuclease IV (EndoIV) families, based on their homology to the two Escherichia coli enzymes. In E.coli the major AP endonuclease is ExoIII (Ec- ExoIII) representing 90% of the cellular AP endonuclease activity, while EndoIV (Ec-EndoIV) accounts for 10% of the total activity [10]. The E.coli AP endonucleases also exhibit additional 39 phosphatase and 39 phosphodiesterase activities in common. These activities are responsible for removing a multitude of blocking groups, including 39 phosphate and 39 phosphoglycolate, that are present at single-stranded breaks in DNA, induced by oxidative agents [11,12]. In addition, Ec-EndoIV and Ec-ExoIII exhibit 39R59 exonuclease activity. Saccharomyces cerevisiae also possesses two AP endonucleases, the Apn1 and Apn2 proteins that represent the EndoIV and the ExoIII family, respectively [13]. However, the major AP endonuclease in this organism is Apn1, that exhibits a strong AP endonuclease activity in yeast cells while the Apn2 protein, is a weak AP endonuclease that exhibits strong 39R59 exonuclease and 39 phosphodiesterase activities [13,14,15,16]. Contrary to the above discovery in budding yeast, in the fission yeast Schizosaccharomyces pombe, Apn2 provides the major AP endonuclease activity while the Apn1 serves only as a back up activity [17]. The human AP endonucleases, Ape1 and Ape2, are both members of the ExoIII family where Ape1 is the PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e71535