DNA building blocks: keeping control of manufacture

Abstract

Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6β2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…Catabolic SAMHD1 and anabolic RNR show striking similarities in their allosteric properties, with allosteric sites binding dNTPs and controlling the activity at the catalytic site. It is intriguing that the same nucleotide, dATP, is a feed-back allosteric inhibitor of RNR, switching off the production of cellular dNTPs, and an efficient allosteric activator of dNTP degradation by SAMHD1 [1,39]. Such coordinated allosteric feedback inhibition of both enzymes would maintain dNTP levels at a proper and balanced level during DNA replication.…”

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

“…T he enzyme ribonucleotide reductase (RNR) is a critical cellular factor for the synthesis and control of the deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA replication and DNA repair (1). Specifically, RNR is responsible for the reduction of the ribonucleotides to the corresponding deoxynucleotides.…”

“…Specifically, RNR is responsible for the reduction of the ribonucleotides to the corresponding deoxynucleotides. In many well-studied cell systems, like the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, or mammalian cells, this reduction takes place at the level of the nucleoside diphosphates (NDP → dNDP), and each of four separate NDPs (ADP, GDP, CDP, and UDP) can serve as RNR substrate (1,2). Following reduction, the dNDPs are converted to the corresponding dNTPs by nucleoside diphosphate kinase (NDK) (3).…”

“…RNRs have been subdivided into several classes, depending on the type of radical used in the catalytic reaction (1,(5)(6)(7). The class Ia RNR, as present in E. coli, yeast, and mammalian cells, employs a tyrosyl radical.…”

“…The activity site is located at the N terminus of the R1 subunit and functions as an "on-off" switch: ATP binding leads to an active enzyme, whereas dATP binding inhibits the enzyme. Hence, by monitoring the ATP/dATP ratio, the enzyme aims to ensure an overall dNTP level that is presumably optimal for DNA replication (1,8).…”