Endonuclease IV Is the Major Apurinic/Apyrimidinic Endonuclease in Mycobacterium tuberculosis and Is Important for Protection against Oxidative Damage

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 3′→5′ exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg2+ and Ca2+ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3′→5′ 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.

DNA Ligase C and Prim-PolC participate in base excision repair in mycobacteria

“…EndoIV lacks a strong 3′-5′ exonuclease activity, present in alternative end-processing enzymes (ExoIII and XthA), and therefore this appears to limit the gap resection to an optimal size for LigC-mediated repair. Consistent with this, it was previously reported that EndoIV, rather than ExoIII, is responsible for vast majority of BER gaps processing in mycobacteria, in contrast to eubacteria where ExoIII plays a more dominant role 30 .…”

Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

“…A variety of studies characterizing extracellular nucleases have focused on the proteins that derived from Streptococcus agalactiae 6 , Streptococcus pyogenes 18 and Staphylococcus aureus 6 19 . Though a previous report had described the intracellular nucleases in M. tuberculosis 20 , no data is currently available regarding extracellular nucleases from the pathogen. In our investigation, the Rv0888 protein was identified as an extracellular nuclease from M. tuberculosis for the first time.…”

Mycobacterium tuberculosis class II apurinic/apyrimidinic‐endonuclease/3′‐5′ exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β‐clamp

“…The bacterial clamp has recently gained attention as a novel target for the development of antibacterials, due to its involvement in a variety of DNA metabolic pathways. Similarly, the XthA homologs from various organisms like E. coli (Cunningham et al, 1986), Neisseria meningitides (Carpenter et al, 2007), Brucella abortus (Hornback and Roop, 2006) and also M. tuberculosis (Sassetti and Rubin, 2003;Puri et al, 2013) have been implicated in bacterial pathogenesis and for helping counter oxidative stress inflicted upon the pathogen by the host. The inhibition of the complex formation between these two important proteins conceivably can affect pathogenesis and survival.…”

“…MtbXthA is a versatile enzyme with AP endonuclease, 3′-5′ exonuclease and 3′ phosphodiesterase activities (Puri et al, 2013;Khanam et al, 2015) like its ExoIII homolog in E. coli (Demple et al, 1986;Shida et al, 1995) and other counterparts from eukaryotic sources (Ishchenko et al, 2003;Burkovics et al, 2006). To evaluate the influence of β-clamp on the different activities of MtbXthA, we carried out the experiments delineated below.…”