Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosisby Sailau Abeldenov, Ibtissam Talhaoui, Dmitry O. Zharkov, Alexander A. Ishchenko, Erlan Ramanculov, Murat Saparbaev, Bekbolat Khassenov

DNA Repair


Cell Biology / Biochemistry / Molecular Biology


Control and prevention of tuberculosis in the United Kingdom: Code of Practice 2000

Joint Tuberculosis Committee of the British Thoracic Society

Access to justice‐the price*

Lord Mackay of Clashfern


Accepted Manuscript

Title: Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis

Author: Sailau Abeldenov Ibtissam Talhaoui Dmitry O.

Zharkov Alexander A. Ishchenko Erlan Ramanculov Murat

Saparbaev Bekbolat Khassenov

PII: S1568-7864(15)00129-9


Reference: DNAREP 2116

To appear in: DNA Repair

Received date: 19-12-2014

Revised date: 19-4-2015

Accepted date: 18-5-2015

Please cite this article as: Sailau Abeldenov, Ibtissam Talhaoui, Dmitry

O.Zharkov, Alexander A.Ishchenko, Erlan Ramanculov, Murat Saparbaev,

Bekbolat Khassenov, Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis, DNA


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Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from

Mycobacterium tuberculosis

Sailau Abeldenova, Ibtissam Talhaouib, Dmitry O. Zharkovc,d, Alexander A. Ishchenkob, Erlan

Ramanculova, Murat Saparbaevb*, Bekbolat Khassenova* aNational Center for Biotechnology, Astana 010000, Kazakhstan. bGroupe «Réparation de l′ADN», Université Paris Sud, Laboratoire «Stabilité Génétique et

Oncogenèse» CNRS, UMR 8200, Gustave Roussy, F-94805 Villejuif Cedex, France. cSB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia dNovosibirsk State University, Novosibirsk 630090, Russia *Corresponding authors. Tel: +33 142115405; fax: +33 142115008 2

Graphical Abstract 3


Mycobacterium AP endonucleases contain 3'-repair phosphodiesterase activities;

Mycobacterium AP endonucleases have very weak AP site cleavage activity;

Mycobacterium homologue of E. coli endonuclease IV contains NIR function;

Both MtbXthA and MtbNfo have optimum activity at pH 6.5;

Expression of MtbXthA and MtbNfo rescue drug sensitivity of E. coli xth nfo mutant. 4


ROS: reactive oxygen species

IR: H2O2, hydrogen peroxide

MMS: methylmethanesulfonate

AP: apurinic/apyrimidinic site

THF: 3-hydroxy-2-hydroxymethyltetrahydrofuran or tetrahydrofuran 3′-P: 3′-phosphate 3′-PA: 3′-phospho α,β-unsaturated aldehyde 3′-THF: 3′-terminal THF residue αdN: alpha-anomeric 2′-deoxynucleosides

DHU: 5,6-dihydrouracil

AP: apurinic/apyrimidinic site αdA: alpha-anomeric 2′-deoxyadenosine

BER: base excision repair

NIR: nucleotide incision repair

Nfo: Escherichia coli endonuclease IV

Xth: Escherichia coli exonuclease III

MtbXthA: Mycobacterium tuberculosis homologue of E. coli exonuclease III

MtbNfo: Mycobacterium tuberculosis homologue of E. coli endonuclease IV

APE1: major human AP endonuclease 1

IPTG: isopropyl β-D-1-thiogalactopyranoside. 5


Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and

DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases:

MtbXthA and MtbNfo, members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and

MtbNfo contain AP endonuclease and 3′5′ exonuclease activities. However, it remains unclear whether these enzymes hold 3′-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3′-repair phosphodiesterase and 3′phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5 mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37°C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60°C. Point mutation analysis showed that

D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3′-blocking sugar-phosphate and 3′phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM = 440 and 1280 M–1·min–1, respectively), while MtbNfo exhibits much lower 3′-repair activities (kcat/KM = 0.26 and 0.65 M–1·min–1, respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees.

Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role in the repair of oxidative DNA damage generated by endogenous and host-imposed factors.

Keywords oxidative DNA damage, Abasic sites, DNA repair, AP endonucleases, 3′-repair phosphodiesterases.

Short title: Mycobacterium AP endonucleases 6 1. Introduction

Cellular DNA encounters two types of endogenous injury: oxidative damage caused by reactive oxygen species (ROS), which comprises oxidized bases, sugars and DNA strand breaks, and spontaneous damage including abasic sites and deaminated bases [1]. Importantly, direct action of

ROS on DNA causes strand breaks, which are a highly cytotoxic type of oxidative damage. Hydroxyl radicals abstract hydrogen atoms from the deoxyribose moiety of nucleotide units in DNA resulting in the release of a free base and an unstable oxidized sugar, which disintegrates with phosphodiester bond breakage. The resulting DNA strand breaks typically bear 5′-phosphate and 3′-blocking groups including 3′-phosphate (3′P) and 3′-phosphoglycolate (3′PGA). If unrepaired, these DNA lesions lead to replication fork collapse and transcription blockage, followed by genome rearrangements and/or cell death [2]. Non-bulky endogenous DNA base lesions and single-strand breaks containing 3′-blocking groups are substrates for two overlapping pathways: base excision repair (BER) and nucleotide incision repair (NIR) [3]. The classic BER pathway involves two sequential excision/incision steps: first, a DNA glycosylase hydrolyses the N-glycosydic bond between the damaged base and the sugar, leaving either an apurinic/apyrimidinic (AP) site or a single-stranded DNA break with a 1-nt gap flanked with a 3′-blocking group and a 5′-phosphate [4-5]. In the second step of BER, an AP endonuclease cleaves 5′ next to AP site leaving a single-stranded DNA break flanked with a 3′hydroxyl group and a 5′-deoxyribose phosphate moiety, or removes 3′-blocking groups in the 1-nt gap to generate proper 3′-hydroxyl termini. Alternatively, in the NIR pathway, an AP endonuclease makes an incision 5′ next to a damaged base and generates a single-strand break with a 5′-dangling modified nucleotide [6]. Non-ligatable DNA strand breaks, generated either directly by ROS or indirectly by oxidative-damage specific DNA glycosylase/AP lyases, are repaired by AP endonucleases that remove 3′-blocking groups by their 3′-repair phosphodiesterase functions to generate 3′-hydroxyl termini that can be used by DNA polymerases to initiate the repair synthesis, as well as by DNA ligases to seal the nicks [7-8].