1. Chapter 24 Nucleotides, Nucleic Acids, and Heredity Nucleotides, Nucleic Acids, and Heredity

2. The Molecules of Heredity Each cell has thousands of different proteins. How do cells know which proteins to synthesize out of 100000s possible amino acid sequences? From the end of the 19th century, biologists suspected that the transmission of hereditary information took place chromosomes in the nucleus, more specifically in structures called chromosomes. genesThe hereditary information was thought to reside in genes within the chromosomes. histonesnucleic acidsChemical analysis of nuclei showed chromosomes are made up largely of proteins called histones and nucleic acids.

3. The Molecules of Heredity deoxyribonucleic acids (DNA)By the 1940s, it became clear that deoxyribonucleic acids (DNA) carry the hereditary information. Other work in the 1940s demonstrated that each gene controls the manufacture of one protein. Thus the expression of a gene in terms of an enzyme protein led to the study of protein synthesis and its control. protein work

4. Structure of DNA and RNA ( based on Nucleic Acids ) Two Kinds in cells: ribonucleic acids (RNA) deoxyribonucleic acids (DNA) RNA & DNA: polymers built from monomers (nucleotides) A nucleotide is composed of: 1. a base, 2. a monosaccharide, 3. a phosphate, (e.g. AMP)

5. 1. Purine/Pyrimidine Bases Base Pairing: DNA: A-T;C-G RNA: A-U;C-G note:

6. Nucleosides (base and sugar) compound that consists of D-ribose or 2-deoxy-D-ribose bonded to a purine or pyrimidine base by a  -N-glycosidic bond.

7. Nucleotides a nucleoside w/ molecule of phosphoric acid esterified with an -OH of the monosaccharide, most commonly either the 3’ or the 5’-OH. ATP- a nucleotide

8. In Summary Nucleoside = Base + Sugar Nucleotide = Base + Sugar + Phosphoric acid Nucleic acid = chain of nucleotides

9. Structure of DNA and RNA - bases arranged in various patterns (like A.A.s for Proteins) GENE  ((protein)) sequence is read from the 5’ end to the 3’ end start finish Primary Structure

10. DNA - 2° Structure the ordered arrangement of nucleic acid strands. the double helix model of DNA 2° structure was proposed by James Watson and Francis Crick in 1953. Double helix: Double helix: 2° structure of DNA in which two polynucleotide strands are coiled around each other in a screw-like fashion. Using Chargaff rules: (A-T; C-G) -X-ray (Franklin, Wilkins) (R. Franklin, 1920-1958) Watson, Crick and Wilkins (Nobel Prize 1962)

11. The DNA Double Helix -Polynucleotide chains run anti-parallel -Bases (hydrophobic) avoid water & stabilize d. helix w/ H-bonds (below)

12. Base Pairing

13. Higher Structure of DNA histones.DNA is coiled around proteins called histones. Histones are rich in the basic amino acids Lys and Arg, whose side chains have a positive charge. The negatively-charged DNA molecules and positively- charged histones attract each other and form units called nucleosomes. Nucleosome:Nucleosome: a core of eight histone molecules around which the DNA helix is wrapped. _+__+_

14. Chromosomes chromatin. Nucleosomes are further condensed into chromatin. chromosomes. Chromatin fibers are organized into loops, and the loops into the bands that provide the superstructure of chromosomes.

15. DNA vs RNA DNA vs RNA 3 differences in structure between DNA & RNA 1. DNA bases/binds A-T, C-G 2. RNA bases/binds, A-U,C-G 2-deoxy-D-riboseSugar in DNA is 2-deoxy-D-ribose; D-ribose.Sugar in RNA it is D-ribose. double strandedDNA is always double stranded; single-stranded.Several kinds of RNA, all of which are single-stranded.

16. RNA- 4 types: 1.Messenger RNA (mRNA) -Carries genetic info f/ DNA to ribosome -Acts as template for protein synthesis 2. Transfer RNA (tRNA) -RNA that transports amino acids to site of protein synthesis (ribosomes) 3. Ribosomal RNA (RNA) -RNA complexed with proteins in ribosomes 4. Ribozymes -Catalytic RNA, with special enzyme functions (e.g. splicing)

17. RNA- 4 types: 1.Messenger RNA (mRNA) -Carries genetic info f/ DNA to ribosome -Acts as template for protein synthesis 2. Transfer RNA (tRNA) -RNA that transports amino acids to site of protein synthesis (ribosomes) Eventually makes a protein to do work (enzymes Modify)

18. Transcription mRNA tRNA rRNA Another perspective, where, what and how

19. Information Transfer

20. Genes, Exons, and Introns Gene: Gene: a segment of DNA that carries a base sequence that directs the synthesis of a particular protein, tRNA, or mRNA. There are many genes in one DNA molecule. In bacteria the gene is continuous. In higher organisms the gene is discontinuous. Exon: Exon: a section of DNA, when transcribed, codes for a protein or RNA. Intron: Intron: a section of DNA or mRNA that does not code for a protein. (intervening sequences, stability, structure etc.

21. Exons and Introns

22. Splicing

23. DNA Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands as templates. origin of replication. 1. DNA double helix unwinds at a specific point called an origin of replication. bidirectional. 2. Polynucleotide chains are synthesized in both directions from the origin of replication; that is, DNA replication is bidirectional. replication forks 3. At each origin of replication, there are two replication forks, points at which new polynucleotide strands are formed. 1. 2. 3.

24. DNA Replication DNA synthesized from 5’ -> 3’ end (from the 3’ -> 5’ direction of the template). leading strandThe leading strand is synthesized continuously in 5’ -> 3’ direction toward the replication fork. lagging strand Okazaki fragmentsThe lagging strand is synthesized semidiscontinuously as a series of Okazaki fragments, also in the 5’ -> 3’ direction, away from the replication fork.

25. DNA ligase.Okazaki fragments (lagging strand) are joined by the enzyme DNA ligase. semiconservative:Replication is semiconservative: each daughter strand contains one template strand and one newly synthesized strand. DNA Replication

27. Replisomes Replisomes are assemblies of “enzyme factories”.

28. Telomers: TTAGGG Somatic cells vs Stem Cells, fetal cells and cancer cells (immortal) Stem cells have telomerase enzyme telomerase enzyme confers immortality to cells Possible organ regeneration etc.

29. DNA Replication 1. Opening up the superstructure. During replication, the very condensed superstructure of chromosomes /Histones are opened by a signal transduction mechanism. One step of this mechanism involves acetylation and deacetylation of key lysine residues. Acetylation removes a positive charge and thus weakens the DNA- histone interactions.

30. DNA Replication 2. Relaxation of higher structures of DNA. Topoisomerases (also called gyrases)Topoisomerases (also called gyrases) facilitate the relaxation of supercoiled DNA by introducing either single strand or double strand breaks in the DNA. Once the supercoiling is relaxed by this break, the broken ends are joined and the topoisomerase diffuses from the location of the replication fork. Moving this way

31. DNA Replication 3. Unwinding the DNA double helix. Replication of DNA starts with unwinding of the double helix. Unwinding can occur at either end or in the middle. helicasesUnwinding proteins called helicases attach themselves to one DNA strand and cause separation of the double helix. The helicases catalyze the hydrolysis of ATP as the DNA strand moves through; the energy of hydrolysis promotes the movement.

32. DNA Replication Primer/primases PrimersPrimers are short oligonucleotides— 4 to 15 nucleotides long. They are required to start the synthesis of both daughter strands. PrimasesPrimases are enzymes that catalyze the synthesis of primers. Primases are placed at about every 50 nucleotides in the lagging strand synthesis.

33. DNA Replication DNA polymerases DNA polymerases are key enzymes in replication. Once the two strands have separated at the replication fork, the nucleotides must be lined up in proper order for DNA synthesis. In the absence of DNA polymerase, alignment is slow. DNA polymerase provides the speed and specificity of alignment. Along lagging (3’ -> 5’) strand, polymerases can synthesize only short fragments, because these enzymes only work from 5’ -> 3’. Okazaki fragments.These short fragments are called Okazaki fragments. DNA ligase.Joining the Okazaki fragments and any remaining nicks is catalyzed by DNA ligase.

34. DNA finger printing Use PCR– amplify small amt. DNA for comparison e.g. paternity suites (2,3,4) e.g. criminal investigations (6,7,8) - Use restriction enzymes to cleave DNA @ spec. sites -Run gel electrophoresis: separate “bands” of varying DNA (smaller go farthest, larger bands migrate least) Applications: Mom b Dad? Crime scene Suspect Match guilty No Match innocent When + match observed (statistically due to chance = 1 in 100 billion!)

35. 25 25-35 © 2006 Thomson Learning, Inc. All rights reserved The viability of cells depends on DNA repair enzymes that can detect, recognize, and repair mutations in DNA. (from UV, mutagens, etc.) DNA Repair e.g. formation of Thymine dimers ~ UV light etc.

36. DNA Repair Base excision repair(BER) Base excision repair (BER): common repair mechanisms. A specific DNA glycosylase recognizes the damaged base. N(1) It catalyzes the hydrolysis of the  -N-glycosidic bond between the incorrect base and its deoxyribose. It then flips the damaged base, completing the excision The sugar-phosphate backbone remains intact. APapapsite(2) At the AP (apurinic or apyrimidinic) site (i.e. w/o a Purine/Pyrimidine) endonucleas created, an endonuclease catalyzes hydrolysis of the backbone See figure 24.12 aP site (1) (2)

37. DNA Repair BER (cont’d) exonuclease(3) An exonuclease liberates the sugar-phosphate unit of the damaged site (4) DNA polymerase inserts the correct nucleotide (5) DNA ligase seals the backbone to complete the repair aP site (5) (4) (3) Damage Nucleoside

38. 25 25-38 © 2006 Thomson Learning, Inc. All rights reserved NERner NER (nucleotide excision repair) removes and repairs up to 24-32 units by a similar mechanism involving a number of repair enzymes DNA Repair (cont.)

39. Cloning Clone: Clone: a genetically identical population. Cloning: Cloning: a process whereby DNA is amplified by inserting it into a host and having the host replicate it along with the host’s own DNA. Polymerase chain reaction (PCR): Polymerase chain reaction (PCR): an automated technique for amplifying DNA using a heat-stable DNA polymerase from a thermophilic bacterium. Steps: 1. Heat (95*C), unwinds DNA, add primer, cool (70*C) replicates 1 st strand.. Etc.. Repeat step 1. See next slide

40. Cloning- PCR

41. from Nucleotides, and Nucleic Acids, to cloned genetics from Nucleotides, and Nucleic Acids, to cloned genetics