Phosphodiester backbone, of A-DNA

Primary structure of nucleic acids The sequence of bases along the pentose-phosphodiester backbone of a DNA or RNA molecule, read from the 5 end to the 3 end. 

The structure showed that the three base-paired stems I, II, and III form type A-DNA helices and that the core contained the two conserved structural domains described in the previous paragraph. A single Me + ion was bound in close proximity to the pro-Rp oxygen of Ag s phosphate and the N7 position of GlO.l. The phosphodiester backbone of the DNA inhibitor strand was splayed... 

Cleavage of nucleic acids refers to a reaction that results in the breakage of bonds in the phosphodiester backbone of a polynucleotide chain. There are two types of reactions resulting in the cleavage of either P-O or C-O bonds in the nucleic acid backboue (see Figure Id). Cleavage of the P-O bond occurs as a result of uucleophilic attack ou the phosphorus atom via either an intermolecular reaction with a H2O molecule hydrolysis) or an intramolecular reactiou involving the ribose 2 -OH transesterification). Of these two reactions, transesterification is specific to RNA (since DNA lacks a 2 -OH moiety) and can be catalyzed by many M- +... 

The enzyme DNA ligase catalyzes the formation of a phosphodiester bond at a break (nick) in the phosphodiester backbone of a duplex DNA molecule. The enzyme from bacteriophage T4 uses the free energy of hydrolysis ATP as the energy source for the formation of the phosphodiester bond. A covalently modihed form of the enzyme in which AMP is bound to a lysine side chain is an intermediate in the reaction. The intermediate is formed by the reaction of E -i- ATP to form E-AMP -i- PPj. In the next step, the AMP is transferred from the enzyme to a phosphate on the DNA to form a pyrophosphate-linked DNA-AMP. In the last step of the reaction, the phosphodiester bond is formed by the free enzyme to seal the nick in the DNA and AMP is released. 

Ribbon model of double-stranded B-DNA. Each ribbon shows the pentose-phosphodiester backbone of a single-stranded DNA molecule.The strands are antiparallel, one running to the left from the 5 end to the 3 end, the other running to the right from the 5 end to the 3 end. Hydrogen bonds are shown by three dotted lines between each G-C base pair and two dotted lines between each A-T base pair. 

The only stereogenic centers of DNA and RNA are found at the sugar carbons, and because the ribose or deoxyribose are enantiomerically pure, natural nucleic acids are isotactic. The P of the phosphodiester backbone of a nucleic acid is not a stereogenic center, but the two 0 groups of a connecting phosphate are diastereotopic. The phosphorus is thus prochi-ral. This has led to the use of labeled phosphates in mechanistic studies, as described with one example in a Connections highlight on the next page. 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Figure 35-1. A segment of one strand of a DNA molecule in which the purine and pyrimidine bases guanine (G), cytosine (C), thymine (T), and adenine (A) are held together by a phosphodiester backbone between 2 -de-oxyribosyl moieties attached to the nucleobases by an W-glycosidic bond. Note that the backbone has a polarity (ie,a direction). Convention dictates that a single-stranded DNA sequence is written in the 5 to 3 direction (ie, pGpCpTpA, where G, C,T, and A represent the four bases and p represents the interconnecting phosphates).

Correct answer = C. Fluoroquinolones, such as ciprofloxacin, inhibit bacterial DNA gyrase—a type II DNA topoisomerase. This enzyme catalyzes the transient breaking and rejoining of the phosphodiester bonds of the DNA backbone, to allow the removal of positive supercoils during DNA replication. The other enzyme activities mentioned are not affected. Primase synthesizes RNA primers, helicase breaks hydrogen bonds in front of the replication fork, DNA polymerase I removes RNA primers, and DNA igase joins Okazaki fragments. 

A restriction endonuclease catalyzes the hydrolysis of the phosphodiester backbone of DNA. Specifically, the bond between the 3 -oxygen atom and the phosphorus atom is broken. The products of this reaction are DNA strands with a free 3 -hydroxyl group and a 5 -phosphoryl group at the cleavage site (Figure 9.33). d his reaction proceeds by nucleophilic attack at the phosphorus atom. We will consider two alternative mechanisms, suggested... 

Although this reaction is in principle quite simple, it is significantly complicated by specific features of the DNA double helix. First, the two strands of the double helix run in opposite directions. Because DNA strand synthesis always proceeds in the 5 -to 3 direction, the DNA replication process must have special mechanisms to accommodate the oppositely directed strands. Second, the two strands of the double helix interact with one another in such a way that the edges of the bases on which the newly synthesized DNA is to be assembled are occupied- Thus, the two strands must be separated from each other so as to generate appropriate templates. Finally, the two strands of the double helix wrap around each other. Thus, strand separation also entails the unwinding of the double helix. This unwinding creates supercoils that must themselves be resolved as replication continues, as described in Section 28.2. We begin with a consideration of the chemistry that underlies the formation of the phosphodiester backbone of newly synthesized DNA. 

At the level of primary structure, several recent experiments have shown the effect of base sequence on the local structure of DNA. A dramatic example is the crystal structure of d(CpG) as determined by Rich and coworkers ( ). This molecule crystallizes in a left-handed double helical form called Z-DNA, which is radically different in its structural properties from the familiar right-handed B-DNA structure. Dickerson and Drew (10) showed in the crystal structure of the dodecanucleotide d(CGCGAATTCGCG) that the local twist angle of a DNA double helix varies with sequence. Deoxyribonuclease I cuts the phosphodiester backbone of the dodecanucleotide preferentially at sites of high twist angle (l 1). From these and other (12,13) experiments we see that the structure of DNA varies with base sequence, and that enzymes are sensitive to these details of structure. 

Prior to the X-ray analysis, the stereochemical course of the hydrolysis of the Sp diastereomer of thymidine 3 -(4-nitrophenyl [ 0, 0]phosphate) 5 -(4-nitrophenyl phosphate) in H2 0 was determined (26). This synthetic oligonucleotide analog is not a good substrate, and the Sp diastereomer of thymidine 3 -[ 0, 0, 0]phosphate 5 -(4-nitrophenyl phosphate) was obtained as product (recall that on double-stranded DNA the oligonucleotide products contain S -phosphate groups). The simplest explanation for this inversion of configuration is that the enzyme catalyzes the direct attack of water on the phosphodiester backbone of double-stranded DNA, presumably by general basic catalysis. 

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