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Mechanisms of Ageing and Development 133 (2012) 138–146
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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. Concluding remarks
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