Section C Properties of Nucleic Acids

1. Section CProperties of Nucleic Acids • C1 Nucleic Acid Structure • C2 Chemical Properties of Nucleic Acids • C3 Thermal Properties of Nucleic Acids • C4 DNA Supercoiling Yang Xu, College of Life Sciences

2. C1 Nucleic Acid Structure • Bases • Nucleosides • Nucleotides • Phosphodiester • DNA double helix Yang Xu, College of Life Sciences

3. NH3 NH3 O O NH3 O Aderine (A) Guanine (G) Cytosine (C) Uracil (U) Thymine (T) Bases • Purines are bicyclic structures, include: • Adenine and guanine • Pyrimidines are monocyclic, include: • Cytosine, thymine and uracil • T or U • The thymine base is replaced by uracil in RNA • Thymine is 5-methyl-uracil Yang Xu, College of Life Sciences

4. 5 Nucleosides • Nucleosides = Base+Sugar, the bases are covalently attached to the 1'-position of a pentose sugar. Nucleotides • Nucleotides = Base + Sugar + phosphates , in the 5'-position of sugar, 1 - 3 phosphates may be attached. Phosphodiester bonds • Covalent linkage of a phosphate group between the 5'-hydroxyl of one ribose and the 3'-hydroxyl of the next. Yang Xu, College of Life Sciences

5. DNA double helix • Discovery: In 1953 DNA double helix structure were deduced by Watson (34y) and Crick (46y) • Structure: • Two chains of DNA are in a right-handed double helix. • The sugar-phosphate backbones are on the outside, and the planar bases in the center of the helix. • Grooves: Between the backbone strands run the major and minor grooves. Yang Xu, College of Life Sciences

6. Base pairing • Base pairs: The strands are joined by hydrogen bonding between the bases on opposite strands, to form base pairs. Yang Xu, College of Life Sciences

7. C2 Chemical Properties of Nucleic Acids • Effect of acid • Effect of alkali Yang Xu, College of Life Sciences

8. Effect of acid for DNA & RNA • Complete hydrolysis • In strong acid and at high temperatures, for example perchloric acid (HClO4) at more than 100 C, • nucleic acids are hydrolyzed completely to their constituents: bases, ribose (or deoxy-ribose) and phosphate. • Partly hydrolysis • In dilute acid, for example at pH 3-4, the most easily hydrolyzed bonds are selectively broken. • Apurinic hydrolysis: The glycosylic bonds attach the purine bases to the ribose backbone, if they are broken the nucleic acid becomes apurinic Yang Xu, College of Life Sciences

9. Effect of alkali for DNA • DNA denaturation: The double-stranded structure of the DNA breaks down; that is the DNA becomes denatured. Effect of alkali for RNA • RNA hydrolysis: In alkali,the hydrolysis of RNA comes about, because of the presence of the 2‘-OH group in RNA, which is participated in the cleavage of the RNA back-bone by intra-molecular attack on the phosphodiester bond. Yang Xu, College of Life Sciences

10. C3 Thermal Properties of Nucleic Acids • Thermal denaturation • Renaturation Yang Xu, College of Life Sciences

11. Thermal denaturation • Increased temperature can cause the thermal denaturation of DNA and RNA: • RNA denatures gradually on heating, but • dsDNA ‘melts’ into single strands at a defined temperature, melting temperature(Tm), • Tm is a function of G+C content of the DNA • Denaturation may be detected by the change in A260. Yang Xu, College of Life Sciences

12. Renaturation • The thermal denaturation of DNA may be reversed by cooling the solution. • The speed of cooling has an influence on the outcome: • Rapid cooling allows only to form dsDNA in local regions, it is not the original dsDNA molecule. • Slow cooling allows the sample fully double-stranded, with the same absorbance as the original dsDNA sample. • The renaturation between different nucleic acid strands is known as hybridization. Yang Xu, College of Life Sciences

13. C4 DNA Supercoiling • Closed-circular DNA • Supercoiling • Topoisomer • Twist and writhe • Energy of supercoiling • Topoisomerases Yang Xu, College of Life Sciences

14. Closed-circular DNA • Many DNA molecules in cells consist of closed-circular double-stranded molecules, for example: • bacterial plasmids; • bacterial chromosomes; • many viral DNA molecules. • This means that: • the two complementary single strands are each joined into circles, and has no free ends; • the molecules are twisted around one another and the two single strands are linked together a number of turns in the molecule. • This turn number is known as the linking number. Yang Xu, College of Life Sciences

15. Supercoiling • Supercoiling: • It is the helix over the helix of dsDNA; • It happens in the closed-circular dsDNA. • Supercoiling direction: • Positive: the twist is in same direction as the double helix; • Negative: the twist is in opposite direction as the helix. • Lk and Lk • Lk : the value for a relaxed closed circle; • Lk: Lk = Lk - Lk, defined as the number of 360 twists introduced before ring closure. It quantifies thelevel of supercoiling. • Example: Most natural DNA is negatively supercoiled • DNA when isolated from cells is commonly negatively (-) supercoiled by around 6 turns per 100 turns, • that is Lk/Lk = -6/100 = -0.06. Yang Xu, College of Life Sciences

16. Positive Negative Yang Xu, College of Life Sciences

17. Topoisomer • The Lk is a topological property of a closed-circular DNA; • The linking number cannot be changed without breaking one or both of the DNA back-bones. • A molecule of a given linking number is known as a topoisomer. • Topoisomers differ from each other only in their linking number. Yang Xu, College of Life Sciences

18. Twist and writhe • Topological changes The conformation of the DNA can be altered while the Lk remains constant (Fig. 2), corresponding to the types of the supercoiling (Lk), the DNA may be: • Completely into writhe (p45 Fig. 2a); • Completely into twist (p45 Fig. 2c); • Common situation is between the two extremes (2b). Yang Xu, College of Life Sciences

19. Twist and writhe • Tw and Wr: • Tw: Twist linking number • Wr: Writhe linking number • Lk = Tw + Wr: • Lk must be an integer, but • Tw and Wr need not. Yang Xu, College of Life Sciences

20. Energy of supercoiling • Torsional stress: • Supercoiling can introduce torsional stress into DNA molecules. Supercoiled DNA hence has a higher energy than relaxed DNA. • Rolesof torsional stress: • For negative supercoiling, this energy makes it easier for the DNA helix to be locally untwisted. • Negative supercoiling may facilitate the processes which require unwinding of the helix, such as transcription initiation or replication. Yang Xu, College of Life Sciences

21. Topoisomerases • Topoisomerases are essential enzymes in all organisms • Being involved in replication, recombination and transcription. • Topoisomerases: The enzymes that regulate the level of supercoiling of DNA molecules are termed topoisomerases • To alter Lk: they break transiently one or both DNA strands; • By attacking a tyrosine residue on a backbone; • There are two classes of topoisomerase: • Type I: breaking one strand of the DNA, and change the Lk in steps of ±1 (p46 Fig. 4a). • Type II: require the hydrolysis of ATP, break both strands of DNA and change Lk in steps of ±2 (p46 Fig. 4b). Yang Xu, College of Life Sciences

22. Yang Xu, College of Life Sciences

23. Segregation Yang Xu, College of Life Sciences

24. That’s all for Section C Yang Xu, College of Life Sciences