ANNOTATION
for the dissertation of Abeldenov Sailau, titled “Role of DNA repair
enzymes in the pathogenesis of tuberculosis” for the Doctor of Philosophy (PhD)
degree in specialty “6D060700 – Biology”
The relevance of the study. In 2013, an estimated by World Health
Organization 9.0 million people developed TB and 1.5 million died from the disease.
For 2013 in the Republic of Kazakhstan, according to World Health Organization,
20000 new cases are revealed. Fight against TB is one of priority problems of health
care of Kazakhstan. TB leads to huge human losses, and also to economic losses of
the country.
TB - a disease caused by Mycobacterium tuberculosis (M.tuberculosis), the
bacteria that infected more than a third of the world population. Treating TB requires
prolonged use of anti-TB drugs. The emergence of multi-drug resistant TB and
extensively drug-resistant TB is a huge risk, as these forms completely bypass all
treatments. Therefore, understanding the mechanism of persistence of M.tuberculosis
within the body is essential for the planning of short and effective treatment of the
disease.
During infection, M.tuberculosis meets a variety of mechanisms of organism
protection. The mechanisms that it meets at an early stage of an infection, probably,
are the most critical for a pathogen survival. It, mainly, makes respiratory explosion
that as a result leads to formation of reactive forms of intermediate compounds of
oxygen and nitrogen. While, the most pathogenic bacteria are eliminated by these
antimicrobic reactions in the infected macrophages, M.tuberculosis evolved
mechanisms for destruction of these forms of protection of an organism, and also
developed an active repair of caused damages.
Once in the body, cellular DNA of M.tuberculosis pathogen is exposed to
reactive oxygen and nitrogen species of intracellular and extracellular origin, which
in turn results in DNA damage. These injuries lead to mutations. Mycobacteria, being
under stress, should maintain the stability of the genome, therefore, the study of DNA
repair systems of pathogen is very important for understanding the mechanisms of
maintaining genetic stability, mutagenesis, and the emergence of multi-drug resistant
strains.
Given the fact that DNA repair systems play an important role in tolerance
against DNA damage caused by nitrogen intermediates and reactive oxygen species,
it is important to study DNA repair system, which may serve as potential targets.
Cellular DNA is constantly exposed to reactive radicals of intracellular or
extracellular origin, often leading to irreversible changes in the genetic map. Such
changes lead to mutations.
Because mycobacteria is constantly exposed to hostile environmental factors,
resulting in formation of damage and modifications of DNA, the genome is protected
by the safety mechanisms of DNA repair. These mechanisms are very important
because they can affect the processes leading to drug resistance and may provide
novel targets for antibiotics.
The research goal and objectives:
The goal of the research is a biochemical characterization of mycobacterial DNA
repair enzymes MtbXthA and MtbNfo of causative agent of tuberculosis
Mycobacterium tuberculosis.
The goal included following objectives:
1. Generation of genetic constructions (vectors) capable to replicate in cells of
Escherichia coli containing Mycobacterium tuberculosis DNA repair genes: nfo/end
and xtha.
2. To get a highly purified recombinant analogues of mycobacterial DNA repair
proteins: MtbNfo and MtbXthA.
3. To determine the binding constant of recombinant proteins MtbNfo and
MtbXthA with radiolabeled DNA oligonucleotides containing unmodified DNA and
DNA with damaged bases using the method of separation of DNA fragments in the
gel.
4. To perform complementation by homologues of Mycobacterium tuberculosis
in determining the sensitivity of mutant Escherichia coli strains, in which genes of
repair proteins to oxidative DNA damage were deleted.
The scientific novelty of the research.
The scientific novelty of the work is for the first time DNA repair enzymes
MtbNfo and MtbXthA of causative agent of tuberculosis Mycobacterium tuberculosis
were biochemically characterized.
For the first time it was shown that in the absence of metal cofactors the
mycobacterial AP endonucleases exhibit very low AP site cleavageactivity.
For the first time it was demonstrated that MtbXthA and MtbNfo possess repair
3'-phosphodiesterase, AP endonuclease, 3 '→ 5' exonuclease and phosphatase
activities.
For the first time we measured the steady-state kinetic parameters of
mycobacterial AP endonuclease-catalyzed repair reactions.
For the first time the mutant forms of MtbXthA and MtbNfo AP endonucleases
were purified with the changing of amino acids in the active sites of the enzymes.
Practical significance of the research. The results of these studies will help to
define the role of DNA repair enzymes in the occurrence of mutations in the genome
of mycobacteria and possibly origin of multidrug resistance in mycobacteria. The
knowledge gained in the course of the dissertation are prerequisites to understanding
the persistence of mycobacteria in the body, the formation of mutant strains of
mycobacteria, which can form the basis for determining good proteins candidates as
drug targets for effective treatment of tuberculosis.
Main conclusions based on research results:
- The mycobacterial AP endonucleases cleave duplex DNA containing a
synthetic AP site;
- It was determined that MtbXthA-catalyzed AP endonuclease activity was
stimulated by Mg2+, Mn2+and Co2+ while MtbNfo activity, by Mg2+and Ca2+and to
lesser extent by Mn2+and Co2+. Furthermore, in the presence of 10 mM MnCl2 both
enzymes exhibit the highest AP endonuclease activity at pH 6.5, whereas at pH 7.5,
high concentration of Mn2+ completely inhibits MtbXthA and MtbNfo-catalyzed AP
site cleavage;
- It was revealed that MtbXthA in addition to AP site cleavage activity, possess a
highly efficient function to remove 3’- blocking sugar-phosphates and 3’-terminal
phosphate remnants from DNA strand breaks. These results demonstrate that
MtbXthA possess highly efficient MtbXthA 3'-phosphatase activity, while MtbNfo is
a bad 3'- phosphatase. Results suggest that both mycobacterial enzymes contain 3’end cleansing activities and that MtbXthA is more efficient 3’-repair
phosphodiesterase than AP endonuclease.
- Mutant mycobacterial AP endonu-cleases, even when present in excess
amounts, do not show anydetectable DNA repair activities as compared to wild-type
MtbXthAand MtbNfo. Importantly, MtbXthA D180N and N182A and
MtbNfoE129Q and H206N mutants concomitantly lost their non-specific3’→5’
exonuclease activity. These results indicatethat D180 and N182 of MtbXthA and
E129 and H206 of MtbNfo are essential for DNA repair activities of Mtb AP
endonucleases.
Publications. The main findings and results of the dissertation were published
in 8 scientific papers, including 1 article in peer-reviewed journal with impact-factor,
cited by Scopus and Thomson Reuters databases, 3 articles in the journals
recommended by the Committee for Control of Education and Science of Ministry of
Education and Science of the Republic of Kazakhstan, 4 in the proceedings of the
republican and international conferences.
Structure of dissertation. Dissertation consists of an introduction, materials
and methods, results and discussion, conclusions, reference sources and appendix.