DNA Structure and Replication: The Blueprint of Life

1. DNA Is the Genetic Material • DNA has a double-helix structure that is made of two strands of polynucleotides. • Each nucleotide consists of a: • Nitrogenous base • Pentose sugar • Phosphate group A Nucleoside Nitrogenous base Phosphate group Pentose sugar A Nucleotide

2. Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Phosphate DNA nucleotide Sugar (deoxyribose) 3 end Guanine (G)

3. In DNA • Two Groups of Nitrogenous bases: • Pyrimidines: • Thymine (T) • Cytosine (C) • Purines: • Adenine (A) • Guanine (G)

4. Sugar and Phosphate Group in DNA

5. 5¢ end Nucleoside Nitrogenous base Phosphate group Pentose sugar Nucleotide 3¢ end Polynucleotide, or nucleic acid

6. Chargaff’s Rules • In the DNA of each species: • The amount of adenine equals the amount of thymine; therefore, the A% = T% in a DNA molecule. • The amount of guanine equals the amount of cytosine; therefore, the G% = C% in a DNA molecule.

7. Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Cytosine (C)

8. 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Key features of DNA structure Partial chemical structure Space-filling model

9. The Basic Principle: Base Pairing to a Template Strand • Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication. • In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules.

10. Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand. • Competing models were the conservative model and the dispersive model

11. A Model of DNA Replication: The Basic Concept The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

12. Getting Started: Origins of Replication • Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”. • A eukaryotic chromosome may have hundreds or even thousands of origins of replication. • At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating.

13. Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand LE 16-12 Replication fork Bubble Two daughter DNA molecules In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome.

14. Elongating a New DNA Strand • Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork. • The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

15. Antiparallel Elongation • The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication. • DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5 to 3direction.

16. Okazaki fragments • Along one template strand of DNA, DNA polymerase can synthesize a complementary strand continuously, called theleading strand, moving toward the replication fork. • To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork. • The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase.

17. 3¢ 5¢ Parental DNA Leading strand 5¢ 3¢ Okazaki fragments Lagging strand LE 16-14 3¢ 5¢ DNA pol III Template strand Leading strand Lagging strand Template strand DNA ligase Overall direction of replication

18. Proteins That Assist DNA Replication-1 • DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end • The initial nucleotide strand is a short one called an RNA or RNA primer • An enzyme called primase can start an RNA primer from scratch • Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately

19. Proteins That Assist DNA Replication-2 • Helicase untwists the double helix and separates the template DNA strands at the replication fork • Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template • Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

20. Proteins That Assist DNA Replication-3 • Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments • DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments

21. Proteins That Assist DNA Replication-4 • DNA polymerase III continuously synthesizes the leading strand and elongates Okazaki fragments by adding nucleotides to 3’ ends. • DNA polymerase I removes primer from the 5 ends of the leading strand and Okazaki fragments, replacing primer with DNA by adding nucleotides to adjacent 3 ends

22. DNA Replication (A Summary) Overall direction of replication Lagging strand Leading strand Origin of replication LE 16-16 Leading strand Lagging strand OVERVIEW DNA pol III Leading strand DNA ligase Replication fork 5¢ DNA pol I 3¢ Primase Lagging strand Parental DNA DNA pol III Primer 3¢ 5¢