Deoxyribonucleotides from ribonucleotides

Enzyme used to produce deoxyribonucleotides from ribonucleotides, its cofactor, and its regulation... 

Which substance is not required for the biosynthesis of deoxyribonucleotides from ribonucleotides ... 

Nucleoside monophosphates are converted to their triphosphates by enzymatic phosphorylation reactions. Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase, an enzyme with novel mechanistic and regulatory characteristics. The thymine nucleotides are derived from dCDP and dUMP. 

All deoxyribonucleotides (used to synthesize DNA) are synthesized from ribonucleotides by the enzyme ribonucleotide reductase, which requires thioredoxin as a cofactor. This enzyme is highly regulated, for example, it is strongly inhibited by dATP—a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase deficiency (see below). 

Thymine deoxyribonucleotides can be generated from ribonucleotides only via UDP. Hence, one must first convert CTP to UDP, then UDP to dUDP, and then to dTTP (see Figure 10.11). 

FIGURE 22-39 Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase. Electrons are transmitted (blue arrows) to the enzyme from NADPH by (a) glutaredoxin or (b) thioredoxin. The sulfide groups in glutaredoxin reductase are contributed by two molecules of bound glutathione (GSH CSSC indicates oxidized glutathione). Note that thioredoxin reductase is a flavoenzyme, with FAD as prosthetic group. 

Nucleotides differ from nucleosides in that the latter do not contain phosphate, so we sometimes refer to nucleotides as nucleoside (mono,di, or tri)-phosphates. For example, adenosine diphosphate is a nucleotide (also called ADP) Deoxyribonucleotides (written with a d ) differ from ribonucleotides in containing deoxyribose as the sugar moiety instead of ribose. In some naming schemes, deoxythymidine nucleotides are written without the d, but the d convention will be used here. 

It should be realized at the outset that all organisms have to possess the capacity to make deoxyribonucleotides firom ribonucleotides. This is the only process which permits the cell to utilize one fraction of the total nucleotides formed de novo in pyrimidine and purine biosynthesis for DNA replication there is no alternative biochemical route producing 2-deoxyribose, its phosphates, or N-glycosides from other molecules (Scheme II). 

The pathways of deoxyribonucleotide synthesis from ribonucleotides are summarized below. The trivial names of the enzymes of ribonucleotide reduction are as follows ribonucleoside diphosphate reductase ribonu-cleoside triphosphate reductase thioredoxin reductase. Enzyme Commission numbers and systematic names have not yet been assigned. 

Initial insight of the role of CSN in cell-cycle control came from the finding that csnl and csn2 deletion S. pomhe strains have an S-phase delay [52]. Interestingly, this effect did not occur in strains missing other CSN subunits. The S-phase delay was caused by the accumulation of the cell-cycle inhibitor Spdl (S-phase delayed 1), which is involved in the misregulation of the ribonucleotide reductase (RNR). RNR catalyzes the production of deoxyribonucleotides for DNA synthesis and... 

The deoxyribonucleotides, except for deoxythymidine nucleotide, are formed from the ribonucleotides by the action of an enzyme complex, which comprises two enzymes, ribonucleoside diphosphate reductase and thioredoxin reductase (Figure 20.11). The removal of a hydroxyl group in the ribose part of the molecule is a reduction reaction, which requires NADPH. This is generated in the pentose phosphate pathway. (Note, this pathway is important in proliferating cells not only for generation... 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

The most important pyrimidine derivatives are those upon which biological organisms depend. Cytosine 1018 and uracil 1019 are found in ribonucleic acid (RNA) in the form of their ribonucleotides, cytidine 1020 and uridine 1021, while in deoxyribonucleic acid (DNA), cytosine and thymine 1022 are found in the form of their 2 -deoxyribonucleotides, 2 -deoxycytidine 1023 and thymidine 1024. 5-Methylcytosine 1025 is also found to a small extent (c. 5%) in human DNA in the form of its 2 -deoxyriboside 1026, and 5-(hydroxymethyl)cytosine-2 -deoxyriboside 1027 has also been detected in smaller amounts <2005CBI1>. Many variants of cytosine and uracil can be found in RNA including orotic acid 1028 in the form of its ribonucleotide orotidine 1029. Other pyrimidine derivatives to have been isolated from various biological sources include 2 -deoxyuridine 1030, alloxan 1031, and toxopyrimidine (pyramine) 1032 (Figure 2). 

Conversion of ribonucleotides to deoxyribonucleotides is an important process that occurs by several pathways, and in one of these, tyrosyl radicals 71 are formed, which serve to generate thiyl radicals from cysteine residues (equation A mechanism for this process has been proposed by... 

Fig. 9 Ribonucleotide reductase mechanism for conversion of ribonucleotides to 2 -deoxyribonucleotides (reproduced from reference 379 with the permission of the American Chemical Society).

Deoxyribonucleotides, the building blocks of DNA, are derived from the corresponding ribonucleotides by direct reduction at the 2 -carbon atom of the D-ribose to form the 2 -deoxy derivative. For example, adenosine diphosphate (ADP) is reduced to 2 -deoxyadenosine... 

The nucleotides described thus far in this chapter all contain ribose (ribonucleotides). The nucleotides required for DNA synthesis, however, are 2 -deoxyribonucleotides, which are produced from ribonucleoside diphosphates by the enzyme ribonucleotide reductase. 

Differences between 5 - 3 and 3 - 5 exonucleases The 5 - 3 exonuclease activity of DNA polymerase I differs from the 3 - 5 exonuclease used by both DNA polymerase I and III in two important ways. First, 5 3 exonuclease can remove one nucleotide at a time from a region of DNA that is properly base-paired. The nucleotides it removes can be either ribonucleotides or deoxyribonucleotides. Second, 5 —>3 exonuclease can also remove groups of altered nucleotides in the 5 —>3 direction, removing from one to ten nucleotides at a time. This ability is important in the repair of some types of damaged DNA. 

Tracer studies with isotopically labeled precursors have shown that both in mammalian tissues and in microorgan-isms, deoxyribonucleotides are formed from corresponding ribonucleotides by replacement of the 2 —OH group with hydrogen. 

The manner in which the reduction of ribonucleotides to deoxyribonucleotides is regulated has been studied with reductases from relatively few species. The enzymes from E. coli and from Novikoff s rat liver tumor have a complex pattern of inhibition and activation (fig. 23.25). ATP activates the reduction of both CDP and UDP. As dTTP is formed by metabolism of both dCDP and dUDP, it activates GDP reduction, and as dGTP accumulates, it activates ADP reduction. Finally, accumulation of dATP causes inhibition of the reduction of all substrates. This regulation is reinforced by dGTP inhibition of the reduction of GDP, UDP, and CDP and by dTTP inhibition of the reduction of the pyrimidine substrates. Because evidence suggests that ribonucleotide reductase may be the rate-limiting step in deoxyribonucleotide synthesis in at least some animal cells, these allosteric effects may be important in controlling deoxyribonucleotide synthesis. 

Many of the enzymes participating in de novo synthesis of deoxyribonucleotide triphosphates, as well as those responsible for interconversion of deoxyribonucleotides, increase in activity when cells prepare for DNA synthesis. The need for increased DNA synthesis occurs under three circumstances (1) when the cell proceeds from the G0, or resting, stage of the cell cycle to the S, or synthetic or replication, stage (fig. 23.26) (2) when it performs repair after extensive DNA damage and (3) after infection of quiescent cells with virus. When cells leave G0, for example, enzymes such as thymidylate synthase and ribonucleotide reductase, increase as well as the corresponding mRNAs. These increases in enzyme amount supplement allosteric controls that increase the activity of each enzyme molecule. Corresponding decreases in amounts of these enzymes and their mRNAs occur when DNA synthesis is completed. 

The biosynthetic pathway to UMP starts from carbamoyl phosphate and results in the synthesis of the pyrimidine orotate, to which ribose phosphate is subsequently attached. CTP is subsequently formed from UTP. Deoxyribonucleotides are formed by reduction of ribonucleotides (diphosphates in most cells). Thy-midylate is formed from dUMP. 

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