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Mechanisms of Ageing and Development 133 (2012) 138–146 Contents lists available at SciVerse ScienceDirect Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev Review Distinct mechanisms of DNA repair in mycobacteria and their implications in attenuation of the pathogen growth Krishna Kurthkoti a, Umesh Varshney a,b,* a b Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India A R T I C L E I N F O A B S T R A C T Article history: Available online 1 October 2011 About a third of the human population is estimated to be infected with Mycobacterium tuberculosis. Emergence of drug resistant strains and the protracted treatment strategies have compelled the scientific community to identify newer drug targets, and to develop newer vaccines. In the host macrophages, the bacterium survives within an environment rich in reactive nitrogen and oxygen species capable of damaging its genome. Therefore, for its successful persistence in the host, the pathogen must need robust DNA repair mechanisms. Analysis of M. tuberculosis genome sequence revealed that it lacks mismatch repair pathway suggesting a greater role for other DNA repair pathways such as the nucleotide excision repair, and base excision repair pathways. In this article, we summarize the outcome of research involving these two repair pathways in mycobacteria focusing primarily on our own efforts. Our findings, using Mycobacterium smegmatis model, suggest that deficiency of various DNA repair functions in single or in combinations severely compromises their DNA repair capacity and attenuates their growth under conditions typically encountered in macrophages. ß 2011 Elsevier Ireland Ltd. All rights reserved. Keywords: Mycobacterium tuberculosis Mycobacterium smegmatis Hypoxia DNA damaging agents 1. Introduction The macrophages internalize various pathogens by phagocytosis and respond to them by generating reactive oxygen and nitrogen species (ROS and RNI), low pH, etc. as part of their innate immune response. Both ROS and RNI can permeate through the cell wall/membrane of the pathogen and serve as important antimicrobial agents (Schlosser-Silverman et al., 2000; Fang, 1997) causing irreversible changes to their biomolecules including DNA. Common damages that occur in DNA are the base modifications, generation of abasic sites and strand breaks (Wink et al., 1991). Inability to rectify such damages is detrimental to the pathogen’s survival in the host. In Salmonella, an intracellular pathogen, deletion of base excision repair (BER) enzymes involved in oxidative damage repair compromised its survival within macrophage cells (Suvarnapunya et al., 2003; Suvarnapunya and Stein, 2005). Mycobacteria constitute an important group of pathogenic bacteria that cause the dreadful diseases of tuberculosis (TB) and * Corresponding author at: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India. Tel.: +91 80 2293 2686; fax: +91 80 2360 2697. E-mail addresses: [email protected], [email protected] (U. Varshney). 0047-6374/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2011.09.003 leprosy. It is estimated that about a third of the human population may be latently infected with Mycobacterium tuberculosis resulting in two million deaths annually (Dye et al., 1999). The biological niche of the pathogenic mycobacteria is the host macrophages. Pathogen’s ability to sustain within such an environment and cause disease has intrigued clinicians and biologists alike. Years of studies have revealed that M. tuberculosis is well equipped to counter DNA damaging agents such as ROS, RNI and the low pH (<3.5) generated by the host immune system (Ehrt and Schnappinger, 2009). Nevertheless, as these agents cause damage to DNA, analysis of how genome integrity is maintained in mycobacteria is an important area of research. In fact, the bacterium must possess robust DNA repair machinery. However, a major DNA repair pathway, the methyl directed mismatch repair pathway is missing in this bacterium (Cole et al., 1998; Springer et al., 2004). And, while the DNA repair machinery is of paramount importance to the pathogen, study of its relevance in pathogenesis has received less attention. We have been interested in the study of nucleotide excision repair (NER), and base excision repair (BER) pathways in mycobacteria. As mycobacteria possess a G+C rich (65%) genome (Cole et al., 1998), BER pathways that repair uracil and 8-oxoG are of particular interest. Here, we review the current status of research on NER and BER pathways in mycobacteria, with emphasis on research findings from our laboratory. General aspects of DNA repair in mycobacteria have been discussed in various other review articles (Mizrahi and Andersen, 1998; Davis K. Kurthkoti, U. Varshney / Mechanisms of Ageing and Development 133 (2012) 138–146 139 and Forse, 2009; Dos Vultos et al., 2009; Kurthkoti and Varshney, 2011). 2. Nucleotide excision DNA repair (NER) Much of our current understanding of NER comes from the pioneering work carried out in Escherichia coli (Rupp et al., 1982). NER was first identified as a pathway that repaired DNA lesions such as thymine dimers resulting from exposure to UV radiation. DNA damages such as abasic sites, DNA cross-links, strand breaks, deamination of bases, etc. generated by ROS and RNI are also used as substrates for NER (Truglio et al., 2006). NER is initiated when UvrA dimer (UvrA2) forms a ternary complex with UvrB [(UvrA2) (UvrB)] and recognizes the damage in DNA in an ATP dependent process (Snowden et al., 1990; Van Houten and Snowden, 1993). The endonuclease, UvrC, is recruited to the site of damage to cleave at 4th or 5th nucleotide downstream and 7th or 8th nucleotide upstream to the damage, leading to excision of 12–13mer oligonucleotide (Sancar and Rupp, 1983). UvrD, a DNA helicase removes the damaged DNA along with the protein complex making way for the DNA polymerase to complete repair synthesis (Fig. 1, Table 1). In human macrophages and lung samples, mycobactria upregulates NER pathway gene transcripts (Graham and ClarkCurtiss, 1999; Rachman et al., 2006). Recently, M. tuberculosis deficient in UvrA was reported to be sensitive to various DNA damaging agents (Rossi et al., 2011). Biochemical analysis of the protein showed that it preferred DNA intermediates with single stranded regions and, similar to other UvrA proteins, the ATPase activity of the M. tuberculosis UvrA was stimulated upon DNA binding. However, its structural determination showed substantial differences from other UvrA proteins (Rossi et al., 2011). Using a transposon mutagenesis screen, another key member of NER, UvrB was also found to be important for bacterial survival. The uvrB mutant of M. tuberculosis showed sensitivity to acidified sodium nitrite (a source of RNI). Further analysis revealed that the mutant was also deficient for its survival within mouse (Darwin et al., 2003; Darwin and Nathan, 2005). Similarly, using Mycobacterium smegmatis model, we observed that targeted disruption of uvrB conferred sensitivity to acidified sodium nitrite. Further, the uvrB mutant displayed high sensitivity to oxidative stress and a severe decrease in survival when subjected to in vitro hypoxia or infected into a murine macrophage cell line (Kurthkoti et al., 2008; our unpublished results). Taken together, these observations suggest NER to be a useful drug target, and the gene knockouts in NER pathway as a means to generate attenuated strains. Recently, a chemical inhibitor 2-(5-amino-1, 3, 4-thiadiazol-2-ylbenzo[f]chromen-3-one (ATBC) that inactivates mycobacterial NER pathway at micromolar concentrations has been reported (Mazloum et al., 2011). It needs to be determined if this molecule or its derivatives can be developed into a therapeutic agent. 3. Base excision repair (BER) BER pathways are initiated by DNA glycosylases that display high degree of specificity for the damaged bases and catalyze their excision by hydrolyzing N–glycosidic bond between the base and the sugar. Excision of damaged bases leads to generation of abasic (AP) sites which are further processed by an AP-endonuclease (e.g., ExoIII), a deoxyribosephosphodiesterase (dRPase, e.g., RecJ, Fpg), a DNA polymerase and DNA ligase to restore the sequence in a short patch (filling in of a single nucleotide) or a long patch (filling in of multiple nucleotides) repair (Fig. 2, Table 1). Some DNA glycosylases such as Fpg (MutM) which excises 8-oxoG from DNA, are bifunctional proteins capable of cleaving the AP-site by their APlyase activity and generating a gap in the sugar phosphate backbone. Fig. 1. Scheme of nucleotide excision repair in eubacteria. A bulky damage in DNA (indicated by star) is recognized by the scanning ternary complex of (UvrA)2UvrB (step I). Identification of damage by UvrB leads to dissociation of (UvrA)2 and recruitment of UvrC (step II). UvrC cleaves DNA at the 5’ and the 3’ sides of the damage (step III) and the action of UvrD helicase leads to removal of UvrB and UvrC proteins along with the cleaved fragment containing the damage (12–13 base oligonucleotide) (step IV). DNA synthesis by DNA polymerase followed by ligation (step V) restores the integrity of DNA. Description M. smegmatis homolog (% identity with E. coli protein)a M. tuberculosis homolog (% identity with E. coli protein)a Remarks/references Ung A family 1 uracil DNA glycosylase. Excises uracil from both single, and double-stranded DNA. Enzyme activity is strongly inhibited by Ugi, a proteinacious inhibitor A family 5 uracil DNA glycosylase. Excises uracil, hypoxanthine, and oxidized pyrimidines from double stranded DNA. Contains iron sulfur cluster and displays thermo-tolerance. Insensitive to inhibition by Ugi Excises alkylated bases e.g., 3-methyladenine MSMEG_2399 (41%) Rv2976c (41%) Ung deficiency in M. smegmatis decreases its survival in macrophage cells. Ung deficiency in M. tuberculosis decreases its survival in mouse(Venkatesh et al., 2003; Sassetti and Rubin, 2003). MSMEG_5031 (UdgB counterpart not present in E. coli) MSMEG_5082 (29%) Rv1259 (UdgB counterpart not present in E. coli) Deficiency of UdgB in M. smegmatis does not cause a significant decrease in survival. However, its deficiency in combination with Ung is synergistic rendering the strain very compromised for growth under RNI and ROS generating conditions (Malshetty et al., 2010; Warner et al., 2010) Rv1210 (29%) AlkA DNA glycosylase has broad substrate specificity and acts on 3-methylpurines and 7-methylpurine Formamidopyrimidine DNA glycosylase excises 8-oxoG paired against C, and shows additional activities of AP lyase and dRPase MSMEG_4925 Rv1317c MSMEG_2419 (33%) Rv2924c (31%) Nei Nei (Endonuclease VIII) acts primarily on oxidized pyrimidines MSMEG_4683 (29%) Rv2464c (26%) MutY Adenine DNA glycosylase acting on oxoG:A base pair in DNA to reverse mis-incorporation of A against 8-oxoG and prevent G:C to T:A transversion. This enzyme also displays AP lyase activity This protein has a DNA dependent ATPase activity. It forms a dimer and binds to UvrB and is known to function as a ‘match maker’ and delivers UvrB to the damaged site In complex with UvrA dimer, UvrB scans DNA to identify damages MSMEG_6083 (34%) Rv3589 (34%) MSMEG_3808 (54%) Rv1638 (53%) TagA may be a major alkylated base excision enzyme in mycobacteria. However, this protein has not been characterized. AlkA is part of AdaA-AlkA composite protein in M. tuberculosis. It lacks alkylbase DNA glycosylase activity but possesses methyltransferase activity (Yang et al., 2011). Deficiency of Fpg in mycobacteria decreases survival in primate model of infection and increases sensitivity to oxidative stress (Jain et al., 2007; Dutta et al., 2010). Mycobacteria have another homolog of Fpg, Fpg2 (Rv0944, MSMEG_5545). Rv2464c (MtuNei1) excises thymine glycol and 5, 6-dihydrouracil (DHU) from DNA and possesses AP lyase activity. It complements E. coli for Fpg or MutY deficiency (Guo et al., 2010). Mycobacteria possess other homologs of the enzyme, Nei2 (Rv3297; MSMEG_1756). Deficiency of MutY in M. smegmatis does not result in any significant phenotypes. However, an increase in C to A mutations is observed. Mycobacterial MutY also removes A paired against G or C (Kurthkoti et al., 2010). Crystal structure of M. tuberculosis UvrA is known. Loss of UvrA increases sensitivity to oxidative stress (Rossi et al., 2011). MSMEG_3816 (53%) Rv1633 (52%) UvrC functions to cleave the DNA strand bound by UvrB, on both sides of the damage Mfd is involved in transcription coupled DNA repair with a bias for damages in the transcribed strand. Mfd binds to the damaged DNA, displaces the RNA polymerase, and recruits Uvr proteins to facilitate repair Following the action of UvrC, UvrD displaces the cleaved DNA, UvrB and UvrC by its helicase activity dUTPase controls excess accumulation of dUTP in cells and prevents its misincorporation in DNA MSMEG_3078 (35%) Rv1420 (36%) MSMEG_5423 (46%) Rv1020 (46%) MSMEG_5534 (36%) Rv0949 (38%) MSMEG_2765 (40%) Rv2697c (36%) MutT MutT hydrolyses 8-oxo-dGTP formed during oxidative stress, and prevents its incorporation in DNA MSMEG_5148 (35%) Rv1160 (27%) Xth Exonuclease III is the major AP endonuclease contributing to cleavage of abasic sites following base excision Performs repair synthesis by filling in the gaps resulting from the action of AP endonucleases or Uvr proteins MSMEG_0829 (27%) Rv0427c (28%) MSMEG_3839 (34%) Rv1629 (32%) MSMEG_2362 (44%) Rv3014c (42%) UdgB TagA AlkA Fpg (MutM) UvrA UvrB UvrC Mfd UvrD Dut DNA polymerase I (PolA) DNA ligase a An NAD+ dependent DNA ligase which restores the phosphodiester bonds following repair synthesis. Action of ligase completes the DNA repair process UvrB deficiency in mycobacteria (M. tuberculosis, M. smegmatis) increases sensitivity to RNI and ROS. Deficiency of UvrB in M. tuberculosis compromises its survival in mouse model of infection (Kurthkoti et al., 2008; Darwin et al., 2003) Identified as an essential gene for survival in mouse model of infection in transposon mutagenesis screen (Sassetti et al., 2003) M. tuberculosis Mfd has been purified and characterized. The C- terminal region of M. tuberculosis Mfd promotes its oligomerization (Prabha et al., 2011) Mycobacteria contain two UvrD proteins; UvrD1 (MSMEG_5534, Rv0949) and UvrD2(MSMEG_1952 and Rv3198c) (Sinha et al., 2007, 2008) M. tuberculosis Dut has been characterized. The protein displays both dUTPase and dCTPase activities. Identified as an essential gene in transposon mutagenesis screen (Helt et al., 2008; Sassetti et al., 2003) There are four MutT like proteins in M. tuberculosis and M. smegmatis (MutT1, 2,3, and 4). Mutants for MutT in M. smegmatis and M. tuberculosis have been generated. MutT2 (Rv1160; MSMEG_5148) which is closest to E. coli displays stronger dCTPase activity (Dos Vultos et al., 2006; Moreland et al., 2009). Other MutTs are: MutT1 (Rv2985, MSMEG_2390); MutT3 (Rv0413, MSMEG_0790) and MutT4 (Rv3908, MSMEG_6927) Biochemical properties of this protein have not been reported from any mycobacteria. Deficiency of PolA shows increased sensitivity to UV and oxidative damage in M. smegmatis (Gordhan et al., 1996). Other polymerases that have been identified in M. tuberculosis and M. smegmatis are DinP (DinB2) Rv3056, MSMEG_2294; DinX (DinB1) Rv1537, MSMEG_3172; DnaE2 Rv3370c, MSMEG_1633 (Kana et al., 2010; Boshoff et al., 2003). Rv3014c is an NAD+ dependent DNA ligase. Mycobacteria possess additional DNA ligases (MSMEG_2277, MSMEG_6302, MSMEG_5570, Rv3062, Rv3731, Rv0938). The other DNA ligases of M. tuberculosis are ATP dependent (Gong et al., 2004). Identity index was calculated using the BLAST tool at www.expasy.org. E. coli protein sequences were used as query to search mycobacterial proteome. K. Kurthkoti, U. Varshney / Mechanisms of Ageing and Development 133 (2012) 138–146 Protein involved in BER and NER 140 Table 1 List of proteins involved in base excision repair and nucleotide excision repair pathways in E. coli and their homologs in M. smegmatis and M. tuberculosis (Davis and Forse, 2009). K. Kurthkoti, U. Varshney / Mechanisms of Ageing and Development 133 (2012) 138–146 Fig. 2. Scheme of base excision repair pathway. A double stranded DNA containing a damaged/modified base (shown in red) is identified by a DNA glycosylase (step I) which hydrolyzes the N-glycosidic bond between the base and the sugar and results in the formation of an abasic (AP) site. Action of AP endonucleases (APE) and deoxyribosephosphodiesterase (dRpase) through (steps II and III) results in the formation of a single nucleotide gap with 3’ OH and 5’ phosphate ends suitable for filling in by DNA polymerase I (step IV) and ligation (step V) to give rise to the repaired DNA. Mycobacteria possess the conserved proteins that participate in BER pathway except that a RecJ homolog has not been identified (Mizrahi and Andersen, 1998; Davis and Forse, 2009). While Fpg is known to possess dRPase activity (Graves et al., 1992; Piersen et al., 2000), it would be important to reconstitute the BER pathway in vitro to understand the role of various repair proteins in mycobacteria (Dianov and Lindahl, 1994; Kumar et al., 2011). 3.1. GO repair pathway Interaction of DNA bases with ROS leads to damages such as strand breaks, inter-strand cross-links and base modifications 141 (Imlay and Linn, 1988). Guanine in DNA is highly sensitive to oxidative damage resulting in generation of 7,8-dihydro-8oxoguanine (8-oxoG) or its derivatives (Fraga et al., 1990; Steenken and Jovanovic, 1997; Farr and Kogoma, 1991; David et al., 2007). Presence of 8-oxoG in the template strand results in misincorporation of A during replication resulting in C to A (or G to T) mutations (Michaels and Miller, 1992; Grollman and Moriya, 1993). To prevent such mutations, organisms maintain an elaborate DNA repair pathway known as GO repair pathway. This pathway involves interplay of two DNA glycosylases and a nucleotide hydrolase. The formamidopyrimidine DNA glycosylase (Fpg or MutM) is the first enzyme in the pathway which excises 8oxoG paired against C. Failure to remove 8-oxoG prior to DNA replication may result in incorporation of A against the damaged base. Interestingly, the other DNA glycosylase, MutY (adenine glycosylase) has a remarkable property of removing the normal base A when paired against 8-oxoG (Au et al., 1989; Lu and Chang, 1988; Tsai-Wu et al., 1992). This action reverses mis-incorporation of A against 8-oxoG and increases chances of incorporation of C against 8-oxoG, and thereby the chances of 8-oxoG removal by Fpg. The third player of the pathway is MutT, a Nudix family nucleotide triphosphate hydrolase (Maki and Sekiguchi, 1992) that degrades 8-oxo-dGTP resulting from oxidative damage of dGTP, thus minimizing the chances of its mis-incorporation into DNA (Fig. 3A, Table 1). Loss of enzyme(s) involved in this pathway affects genomic stability (Chang et al., 2001; Parker et al., 2003). In Pseudomonas (which possesses G+C rich genome) deficiency in GO repair pathway results in increased mutations and sensitivity to the oxidizing agents (Sanders et al., 2009). Mycobacteria also possess G+C rich genomes and home into macrophages making guanines in DNA vulnerable to the oxidative damages. Therefore, to assess the role of GO repair pathway in mycobacteria, we generated Fpg (MSMEG_2419), and MutY (MSMEG_6083) deficient strains of M. smegmatis by gene knockout methods (Jain et al., 2007; Kurthkoti et al., 2010). Loss of Fpg increased the mutation frequency by 3 fold and sensitized the strain to oxidative damage. However, the survival defects were not as severe as observed for the uvrB strain (Kurthkoti et al., 2008). Analysis of the mutation spectrum in the rifampicin resistance determining region of the rpoB gene resulting in rifampicin resistance to the Fpg deficient strain revealed an unexpectedly high incidence of C to G mutations during oxidative stress. Incorporation of nucleotides against 8-oxoG (in a DNA oligomer) using cell-free extracts from M. smegmatis fpg strain revealed mis-incorporation of G against 8-oxoG in the template with a high preference in contrast to the cell-free extract of E. coli wherein mis-incorporation of A was predominant. While these studies provided a rationale for the G bias of the mutations in Fpg deficient strains, the identity and detailed biochemical properties of the error prone DNA polymerase (s) responsible for such mutations are not known. Similar studies using other M. smegmatis strains showed that MutY (MSMEG_6083, Rv3589) deficiency did not lead to any appreciable increase in either the mutation rate or the sensitivity to hydrogen peroxide (Kurthkoti et al., 2010). However, the analysis of mutation spectrum revealed occurrence of expected C to A mutation. In addition, there was a considerable increase in A to G and A to C mutations. Analysis of the substrate specificities of the major Fpg in M. smegmatis (MSMEG_2419) and M. tuberculosis (Rv2924c) revealed that it excises 8-oxoG, when paired against C, G or T but not A (Jain et al., 2007; Guo et al., 2010; Olsen et al., 2009). Additionally, like other Fpg proteins the mycobacterial Fpg possesses formamidopyrimidine (faPy) DNA glycosylase and AP-lyase activities. The substrate specificity analysis of M. smegmatis and M. tuberculosis MutY showed that it excises A against 8-oxoG. However, detectable activity of G and T excision from the 8-oxoG:G and 142 K. Kurthkoti, U. Varshney / Mechanisms of Ageing and Development 133 (2012) 138–146 Fig. 3. (A) General scheme of GO repair pathway. Oxidized guanine (8-oxoG) arising in DNA due to oxidative stress is removed by Fpg which upon action by DNA polymerase followed by ligation restores the genetic information. Replication of DNA against 8-oxoG prior to its excision by Fpg could lead to mis-incorporation of A. Adenine DNA glycosylase (MutY) catalyzes the removal of A from the 8-oxoG:A pair and increases the chances of incorporation of the correct base C, and offers another chance to Fpg to remove 8-oxoG. Inability to correct the error will result in C to A or G to T transversion mutation (as indicated in light purple oval). (B) Distinctive aspects of GO repair pathway in mycobacteria. The overall mechanism of GO repair remains similar to those seen in other eubacteria. The 8-oxoG damage is recognized and removed by Fpg (steps 1 and 2) and repaired to yield wild-type sequence (step 3). Replication prior to repair by Fpg could lead to incorporation of either G or A (step 4) followed by their excision by MutY (step 5) and filling of C (step 6, left) could make it a substrate for Fpg (step 7), and filling with G or A (step 6, right) for MutY (step 8). However, failure to repair by MutY prior to replication could fix the mutations (step 9). The lower part of the figure shows how deficiency of MutT could lead to misincorporation of 8-oxoG against G, A and C (shown by X) and lead to a variety of mutations on account of MutY, Fpg or incorporation of incorrect base against 8-oxoG during replication (steps 10–16). For further details see Jain et al. (2007) and Kurthkoti et al. (2010). K. Kurthkoti, U. Varshney / Mechanisms of Ageing and Development 133 (2012) 138–146 8-oxoG:T pairs was also seen (Jain et al., 2007; Kurthkoti et al., 2010). Interestingly, besides the major MutY and Fpg proteins, mycobacteria possess Nei (Nei1, Rv2464c), and Nei2 (Rv3297) which rescue the mutator phenotype of E. coli strains deficient in Fpg or MutY. In addition, in M. tuberculosis, another Fpg, Fpg2, has also been characterized which may be a nonfunctional Fpg as it lacks the highly conserved N-terminal proline residue that forms a part of the catalytic centre (Sidorenko et al., 2008; Guo et al., 2010). The other arm of the GO repair pathway involves MutT, which hydrolyzes 8-oxo-dGTP and prevents its mis-incorporation in DNA. The M. tuberculosis genome revealed the presence of four MutT like (MutT1, 2, 3 and 4) proteins (Cole et al., 1998). Generation of MutT deficient strains in M. tuberculosis and M. smegmatis resulted in increased mutation frequency to different extents, and the biochemical analysis of the partially purified proteins indicated that the MutTs had differential substrate specificities (Dos Vultos et al., 2006). It was reported that the M. tuberculosis MutT2 which resembles E. coli MutT, displays dCTPase activity (Moreland et al., 2009). Currently, the nature of the other MutTs is not entirely known. Detailed biochemical analyses may reveal the physiological roles of MutTs in mycobacteria. Based on the distinctive properties of the mycobacterial DNA polymerase(s) in incorporating 8-oxoG or a base against 8-oxoG in DNA and, substrate specificities of Fpg, MutY and MutT, deficiencies in GO repair pathway in mycobacteria, may lead to a distinctive mutation spectrum (Jain et al., 2007; Kurthkoti et al., 2010; Fig. 3B). Importantly, the presence of multiple homologs of Fpg, MutY and MutT proteins in mycobacteria indicates that the repair of oxidative damages is critical for the success of M. tuberculosis within macrophage. 3.2. Uracil excision repair pathway Uracil base in DNA, arises either from its incorporation by DNA polymerase or as a consequence of the deamination of the 4-amino group of the resident cytosines (Friedberg et al., 1995). The fact that pathogenic mycobacteria establish themselves in macrophages, the frequency of cytosine to uracil conversion in the G+C rich genome of mycobacteria is likely to be quite high because of the generation of RNI and ROS by the host cell. Accumulation of uracils in the genome is known to affect the viability of organisms (Gadsden et al., 1993; Taylor and Weiss, 1982). Direct incorporation of uracil in DNA by DNA polymerases is minimized by the presence of dUTPase (an enzyme which hydrolyzes dUTP and keeps a low intracellular pool of dUTP) (Tye et al., 1978; Vertessy and Toth, 2009). Interestingly, the dUTPase (encoded by the dut gene, MSMEG_2765, Rv2697c) in mycobacteria possesses additional activity of dCTPase (Helt et al., 2008). The dut gene has been shown to be essential in a transposon mutagenesis screen (Sassetti et al., 2003). The excision repair of uracils in DNA is carried out by uracil DNA glycosylase (Ung), a highly proficient enzyme (Lindahl et al., 1977). Mycobacterial Ung, like other Ung proteins, excises uracil from both the single stranded and double stranded DNA (Purnapatre and Varshney, 1998). Interestingly, while E. coli Ung is inefficient in excising uracils from the loop substrates the mycobacterial Ung is efficient in utilizing such substrates. However, like its counterparts from other organisms, the mycobacterial Ung is sensitive to inhibition by the Bacillus subtilis phage early gene protein, Ugi (Purnapatre and Varshney, 1998; Acharya et al., 2003). Determination of the three dimensional structure of M. tuberculosis Ung showed that while its overall structure is similar to Ung proteins from other sources, its N- and C-terminal tails exhibit high variability and its DNA-binding region possesses higher proportion of arginyl residues (Kaushal et al., 2008). Besides Ung, mycobacteria possess an additional protein, UdgB, which also excises uracil from DNA 143 (Srinath et al., 2007). However, UdgB utilizes only the double stranded DNA substrates, and while the Ung (MSMEG_2399, Rv2976c) is highly specific for uracil, UdgB (MSMEG_5031, Rv1259) has broad substrate specificity, and excises even hypoxanthine and ethenocytosine (generated during RNI and oxidative stresses, respectively, Guillet and Boiteux, 2002; Mamun and Humayun, 2006). UdgB is a thermotolerant Fe–S cluster protein (Srinath et al., 2007). The need for a thermotolerant uracil DNA glycosylase in M. tuberculosis which survives in the host at a physiological temperature of 37 8C is perplexing. Interestingly, our studies show that UdgB has high affinity for AP-sites in DNA. As APsites are highly cytotoxic and mutagenic, an additional role of UdgB could be to protect the AP-sites. Deficiency of Ung in M. smegmatis leads to an increase in mutation rate of 9 fold, and compromised growth under the conditions of in vitro hypoxia as well as those that mimic ROS and RNI stress including mouse macrophages (Venkatesh et al., 2003; Kurthkoti et al., 2008). Subsequently, in a transposon mutagenesis screen, it was observed that mutation in ung resulted in elimination of M. tuberculosis in a mouse model of infection (Sassetti et al., 2003). To address the physiological importance of UdgB and its role as a possible backup for Ung, we and others generated knockout strains of M. smegmatis (Wanner et al., 2009; Malshetty et al., 2010). Analysis of the mutant deficient in UdgB alone revealed a minor increase of 2 fold in mutation rates and a minor effect of the acidified nitrite or peroxide on its growth. Further, consistent with its in vitro substrate specificity (Srinath et al., 2007), the mutation spectrum analysis showed that in addition to C to T, A to G mutations were equally prominent in the udgB strain suggesting that UdgB excised not only U but also hypoxanthine (deamination product of A). More importantly, we observed that udgB mutation in ung background has a synergistic effect. It showed an enhancement in mutation rate to 19 fold compared to the rates of ung or udgB which were 9 and 2 fold, respectively (Kurthkoti et al., 2008; Malshetty et al., 2010). Also, the double mutant was severely compromised for growth in the presence of hydrogen peroxide or the acidified nitrite. It would be important to generate similar mutations in M. tuberculosis and analyze their impact on the pathogen’s growth under various conditions. 4. DNA repair during hypoxia Following infection with M. tuberculosis, the host mounts an immune response to contain its spread. A characteristic feature of the TB pathology is the formation of granuloma, a multicellular structure formed by the host to restrict the tubercle bacilli (Dannenberg and Rook, 1994). Within the granuloma, the bacterium experiences an anaerobic environment and is assaulted by RNI and ROS generated by the surrounding macrophages. In spite of such a host response, the bacterium sustains itself in a state generally defined as the ‘latent’, ‘dormant’ or the persistent state. However, under the conditions of compromised host immunity it reactivates to cause clinical TB (Flynn and Chan, 2001; Selwyn et al., 1989). Aspects of DNA repair in the ‘dormancy’ state of bacterium have not been studied. In the mammalian systems hypoxia is known to cause DNA damages such as base modifications and strand breaks (Moller et al., 2001; Grishko et al., 2001). Animal models established to study mycobacteria in granuloma (Via et al., 2008; Kesavan et al., 2009) are limiting in providing sufficient material to carry out biochemical analyses. As hypoxia is one of the driving forces that facilitate the bacterium to persist within the granuloma, an in vitro model for the persistent state of the pathogen (M. tuberculosis) was established (Wayne and Sohaskey, 2001). Subsequently, the Wayne’s model of hypoxia was adapted for M. smegmatis (Dick et al., 1998; Mayuri et al., 144 K. Kurthkoti, U. Varshney / Mechanisms of Ageing and Development 133 (2012) 138–146 During the last decade, steady progress in the field of mycobacterial DNA repair has identified several processes that are extremely important for survival within the host. The uvrB mutant of mycobacteria, has been shown to be severely compromised in survival within the host (Darwin et al., 2003; Darwin and Nathan, 2005). Members of uracil excision repair pathway are equally important as transposon mutants of dut and ung showed reduced survival in mouse model of infection (Sassetti and Rubin, 2003). In a more recent study, it was reported that members of oxidative DNA damage repair pathway such as fpg and nei are also important for bacterial survival within the primate model of infection (Dutta et al., 2010). Screening of chemical inhibitors for important BER pathways would be an important step to exploit DNA repair system as a potential therapeutic target (Jiang et al., 2004, 2005; Huang et al., 2009). Analysis of the single nucleotide polymorphism (SNP) in M. tuberculosis strains from across the world revealed higher number of SNPs in the genes participating in recombination, repair and replication processes than those participating in house-keeping functions. However, it unclear if such changes affect bacterial survival or promote evolution of antibiotic resistance (Dos Vultos et al., 2008). A study by Boshoff et al. (2003) showed that M. tuberculosis DnaE2, a DNA polymerase belonging to class C family is not only required for bacterial survival within host but also for induction of mutagenesis leading to resistance to antibiotics. The promoter region of dnaE2 contains an SOS box and is strongly induced upon DNA damage by physiological agents such as hydrogen peroxide. In a recent report, Warner et al. (2010) demonstrated that DnaE2, ImuA’ and ImuB physically interact and deletion of ImuA’ or ImuB in mycobacteria resulted in reduction in the UV induced mutagenesis even in the presence of DnaE2. Thus, DnaE2 requires the support of ImuB to bring out its mutagenic effect. It is now becoming clear that the proteins involved in DNA repair may aid in the success of pathogen in different ways, by repairing damaged DNA and facilitating mutagenesis. The latter effect is a major concern as it directly influences emergence of drug resistant variants. A detailed understanding of the different players, which are involved in mutagenesis can provide insights into the mechanism of drug resistance and allow better management of available therapeutics. resistant TB strains has reached alarming proportions requiring new drugs for therapy. Thus, there is a major thrust to identify pathways important in M. tuberculosis that can be exploited as potential targets for developing therapeutics or attenuated strains. Availability of genome sequence of M. tuberculosis (Cole et al., 1998) and that of a nonpathogenic saprophytic counterpart M. smegmatis (The Institute for Genomics Research, www.tigr.org) has provided a major boost to the TB research. Recent studies in the field of bacterial physiology have identified several pathways necessary for survival within the host. Such findings are important to generate useful knockout strains of M. tuberculosis. Many of the mutant strains generated showed defects in survival or virulence (McKinney et al., 2000; Sambandamurthy and Jacobs, 2005; Smith et al., 2001; Brzostek et al., 2007). Though the genome encodes for numerous proteins involved in lipid metabolism there are surprises when it comes to DNA repair. The bacterium lacks the highly conserved mismatch repair pathway. Further, it has been shown that inactivation of recA in M. bovis BCG resulted in increased sensitivity to various DNA damaging agents, but it did not affect its in vitro dormancy response or survival in a mouse infection model (Sander et al., 2001). It is also noteworthy that induction of several DNA repair genes following DNA damage occurs independent of RecA (Rand et al., 2003). The expression of other components of recombination such as RecB and RecC and RuvABC and RecG that resolve Holliday junction, have been shown to be up-regulated following infection (Rachman et al., 2006; Schnappinger et al., 2003). The recombination pathway may be exploited by bacteria to exchange damaged DNA following RNI and ROS attack from the host. Therefore, targeting such components of recombination may provide bacterial strains with reduced survival within the host. Considering the fact that DNA repair pathways play an important role in tolerance of RNI and ROS generated damages in DNA, it is important to explore if the DNA repair pathways can serve as potential targets. Interestingly, there is a high degree of conservation between the DNA repair enzymes in M. smegmatis and M. tuberculosis. And, considering M. smegmatis is a nonpathogen, it provides a useful model to obtain the first information on the distinctive aspects of DNA repair in M. tuberculosis. As described, we studied the impact of deletion of several DNA repair genes (ung, uvrB, fpg, mutY, and udgB) in M. smegmatis. The uvrB strain was observed to be severely compromised for its growth under the commonly encountered DNA damaging conditions of acidified nitrite or hydrogen peroxide, and the Wayne’s model of hypoxic growth (Kurthkoti et al., 2008). While the ung strain was also compromised for growth under such conditions, the growth defect was severe when the ung and udgB mutations were combined (Malshetty et al., 2010). In yet another aspect of our studies, when we subjected M. smegmatis and M. tuberculosis to in vitro hypoxia, expression of a number of DNA repair genes was down-regulated. Further, using M. smegmatis model, we observed that hypoxia specific mis-expression of ung led to its reduced survival (Kurthkoti and Varshney, 2010). The impact of hypoxia specific mis-expression of DNA repair genes in M. tuberculosis is currently not known. However, such studies in M. tuberculosis should prove useful to engineer attenuated strains. 6. 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