11 February 2015

Error Correction Award

In a healthy body – healthy DNA

Andrey Sobolevsky, "Science in Siberia"Nikita Aleksandrovich Kuznetsov, Candidate of Chemical Sciences, Senior researcher at the Institute of Chemical Biology and Fundamental Medicine SB RAS, awarded the Russian President's Prize for young scientists in the field of science and innovation, tells about the essence of his work.

Photo from the personal archive of N.A. Kuznetsov

– All living organisms contain a carrier of genetic information. In humans, it is represented in the form of 46 chromosomes: the main function of the genome is the storage and subsequent transmission of all body codes. The main task set by nature itself is to keep this information unchanged. To do this, there are a number of enzymatic systems that fight DNA damage. Damage can occur spontaneously, as a result of a number of metabolic processes, and under the influence of external factors – ultraviolet radiation, radiation, harmful chemicals and the like.

The task of enzymatic DNA repair systems is to quickly and efficiently find damage and eliminate them. Our work is aimed precisely at understanding, with all the nuances, how the mechanisms of reparation work. If you focus on a person, then he has about 3 billion DNA bases in his genome. Even if the body is in ideal conditions, 10-20 thousand damages spontaneously occur within 24 hours. One can imagine how complex and subtle this mechanism is: the search for even 10 thousand elements in a three-billion set! Note that it contains information about the enzymes themselves involved in the repair process, and if damage occurs in these areas, then very, very sad consequences occur for the body…

Any organism is programmed to maintain the integrity and immutability of genetic information, therefore, repair enzymes must always be in the right amount and in "working condition". The most important thing for us and our colleagues is to understand how these enzymes find damage, how they "distinguish" damaged DNA elements from normal ones. Another aspect of the study of repair mechanisms is related to medicine. For example, in the treatment of oncological diseases, chemotherapy is widely used, which includes the intake of substances aimed precisely at DNA damage – as a rule, alkylating agents. DNA damage also occurs with another common therapy, radiation. If the patient's repair system works well, then its enzymes will find and correct such damage, objectively interfering with the action of oncolytic therapy).

As you understand, there are two main reasons to study the mechanism of enzymatic DNA repair. First, knowledge of the mechanism of this process in a healthy person will allow us to create methods for quickly determining the activity of DNA repair enzymes that can be used to determine the repair status of an organism. In the future, this parameter of the organism can be used as a new criterion for the suitability of people exposed to increased "genomic loads", for example, astronauts, submariners, aircraft pilots, personnel of nuclear and chemical enterprises, etc. On the other hand, understanding the mechanism of finding and eliminating damage, you can try to reduce the activity of the enzymes involved in it, when necessary (in the case of the same chemotherapy).

My colleagues and I are investigating a number of specific DNA repair enzymes that find damage and trigger an enzymatic cycle of reactions that lead to the replacement of damaged nucleotides with natural ones. It is known that such enzymes as DNA glycosylases (in humans there are 11 DNA glycosylases that remove various types of damage from DNA), AP-endonuclease, repair DNA polymerases and DNA ligases take part in this cycle. Our research is primarily related to DNA glycosylases and AP-endonuclease.

In our work we use a number of physico-chemical and biological methods. The methods of genetic engineering and mutant analysis allow us to create expression vectors, to carry out the development and purification of enzymes of interest to us. The use of mutant forms of enzymes, when a new amino acid is introduced into a certain place of the protein instead of the "native" one, allows us to establish the role of a specific amino acid residue in the processes of DNA binding, recognition of a damaged nucleotide and catalytic reactions. To register various stages of the enzymatic process, prestationary kinetics methods are used, which allow registering individual stages of the enzymatic process by changing the fluorescence intensity of tryptophan residues in enzymes or fluorescent groups chemically embedded in model DNA duplexes.

We have two stopped jet spectrometers in our laboratory that allow us to mix two solutions quickly, in 1 millisecond. One of these solutions contains the enzyme under study, the other contains a model DNA duplex containing a certain type of damage. In the process of enzymatic "recognition" of the damaged base, it is "turned out" from the DNA duplex to the active center, and several amino acid residues of the enzyme are embedded in the space formed in the DNA. After that, the final adjustment of the structures of the interacting molecules takes place in order to form a catalytically active complex, and a chemical reaction takes place. The complexity and multistage nature of the processes under study requires the involvement of specialized mathematical calculations for processing and analyzing the experimental data obtained.

The result of the study is the molecular kinetic mechanism of the process of searching and removing the damaged nucleotide. Based on this mechanism, it is possible to assess the prospects and create a way to influence the catalytic activity of DNA repair enzymes. It should be noted that based on the data we have obtained, we have developed and patented methods for determining the activity of two important DNA repair enzymes in humans – 8-oxoguanine-DNA glycosylase and AP-endonuclease. And also a compound was found and patented, currently the only one in the world that is an inhibitor of the enzyme 8-oxoguanine-human DNA glycosylase.

All the work I have described is being carried out in the laboratory of biopolymer modification research led by Olga Semenovna Fedorova, Doctor of Chemical Sciences, my supervisor since my student days.

As a rule, each new student of the laboratory gets a new object of research – an unexplored DNA repair enzyme. I was also no exception, during my graduation practice I conducted an analysis of a bacterial enzyme – formamidopyrimidine-DNA-glycosylase. The PhD thesis was already devoted to two enzymes.

The cycle of works, highly appreciated in the form of the award of the President of the Russian Federation, covers more than a dozen enzymes.

Portal "Eternal youth" http://vechnayamolodost.ru11.02.2015

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