My research area is structural studies of enzymes involved in the synthesis of DNA precursors. These enzymes are of special interest not only because they produce the building blocks of the DNA, but also because they play an important role in anti-tumour and anti-viral therapy. The method I use is X-ray crystallography.

Two ways of dNTP synthesis

The precursors of DNA, deoxyribonucleoside triphosphates, dNTPs, are synthesized in two ways.  One way is through recycling of deoxyribonucleosides (dNs) from food or degraded cells. This process is refered to as salvage pathway. There is a rather big group of enzymes involved in salvage pathway, with big diversity among species. Their common name is deoxynucleoside kinases, DNKs. The other way to produce dNTPs is through the de novo pathway where ribonucleotides (the precursors of RNA) are reduced to form deoxyribonucleotides. The key enzyme here is ridonucleotide reductase, RNR.

The transport of dNs into the cytosol takes place via carrier membrane proteins. These carrier proteins can transport the dNs both into and out of the cell. There is no transport system for nucleotides, which means that once a nucleoside has been phosphorylated it becomes trapped inside the cell and ready to enter the salvage pathway. Inside the cell dNs are phosphorylated in three steps by three unique enzymes, starting with transformation of deoxyribonucleosides to deoxynucleoside monophosphates (dNMPs), continuing to diphosphates (dNDPs), and ending with the final DNA-precursors, dNTPs. The first phosphorylation of the 5’OH group of the deoxynucleoside to a deoxynucleoside monophosphate may be considered as the rate-limiting step. This is because after this initial phosphorylation the monophosphates become trapped inside the cell and can be further phosphorylated in the salvage pathway.

The first phosphoryl transfer from ATP to form dNMP is catalyzed by deoxynucleoside kinases. There are four deoxyribonucleoside-specific kinases in mammalian cells. Two of them, deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1), are found in the cytosol, whereas the remaining two, deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), are mitochondrial enzymes. The enzymes have partly overlapping substrate specificity. dCK has high substrate specificity for dC as well as the purines dG and dA, dGK phosphorylates purine deoxynucleosides, with the highest affinity for dG. TK1 phosphorylates thymidine (dThd) whereas TK2 has the highest specificity for dC. Interestingly, the fruit fly Drosophila melanogster has only one multisubstrate deoxyribonucleoside kinase with high affinity for all natural deoxyribonucleosides.

Apart from being key enzymes in synthesis of dNTPs, the activity and specificity of these enzymes also play an important role in anti-tumour and anti-viral therapy where nucleoside analogues are used. In order to compete with endogenous DNA precursors these compounds must be activated by nucleoside kinases.

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In contrast to salvage pathway, where dNTPs are made from recycling of degraded cells, in de novo pathway the dNTPs are synthesised from scratch. Small compounds such as amino acids, tetrahydrofolate derivatives, NH₄⁺, CO₂ and 5-ribosyl-1-pyrophosphate build up ribonucleotides that futheron are reduced to deoxyribonucleotides. This reaction is catalyzed by ribonucleoside reductase, RNR. RNR reduces all the four main ribonucleotides to the corresponding deoxyribonucleotides, thus it is an essential enzyme in DNA synthesis and indispensable for survival of all living organisms.

The reaction catalysed by RNR is a chemically complexed reaction performed with help of organic free redicals, which are stored by the enzyme until needed. An important elemt in the catalysis is the control of the free radicals. The RNR activity is cell cycle regulated. It is also allosterically regulated in the way where triphosphate nucleotides regulate the substrate specificity such that a balanced supply of the different deoxynucleotides is present during DNA synthesis. Moreover, the overall enzymatic activity is regulated by ATP (activated) and dATP (feed back inhibited at high concentrations).

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