DNA REPLICATION

1. DNA REPLICATION Mader Biology Chapter 13

2. Review: Structure of DNA • Monomer = Nucleotides • Sugar, phosphate, nitrogenous base • A-T, C-G • Backbone: Sugar and Phosphate • “steps” of ladder: bases

3. Helical Structure

4. DNA Replication • Semiconservative • Template strand copied and becomes half of new strand • 2 new complete double helices each with 1 old and 1 new strand

5. Helicase • Helicase is the enzyme that opens double helix and helps it uncoil

6. Single-strand binding proteins (SSBP) • Keep strands separated – large amount of this protein required

7. DNA Polymerase • An enzyme that catalyzes the addition of a nucleotide to the DNA chain • 5’–3’ direction • (read: 5 prime to 3 prime)

8. DNA Polymerase • Bidirectional synthesis of the DNA double helix • Corrects mistaken base pairings • Requires an established polymer (small RNA primer) before addition of more nucleotides

9. How is DNA Synthesized? • Called the leading strand • DNA polymerase reads 3’  5’ along the leading strand from the RNA primer • Synthesis proceeds 5’  3’ with respect to the new daughter strand • Remember how the nucleotides are added!!!!! 5’  3’

10. Starting Synthesis • DNA polymerase can only ADD nucleotides to a growing polymer • Another enzyme, primase, synthesizes a short RNA chain called a primer • DNA/RNA hybrid for this short stretch • Base pairing rules followed (BUT A-U) • Later removed, replaced by DNA and the backbone is sealed (ligated)

11. Okazaki Fragments • Leading Strand: Simple addition of primer • 5’  3’ primer then DNA • Lagging Strand: Many primers are needed • Makes many small strands • Called Okazaki fragments

12. Removal of Primers • Other enzymes needed to remove the primers • DNA ligase – seals the sugar-phosphate backbone by creating phosphodiester bond

13. Mistakes during Replication • Base pairing rules must be maintained • Mistake = genome mutation, may have consequence on daughter cells • If wrong nucleotide is included • Polymerase uses its proofreading ability to cleave the phosphodiester bond of improper nucleotide • Activity 3’  5’ • And then adds correct nucleotide and proceeds down the chain again in the 5’  3’ direction

14. Proofreading

15. DNA Repair • For the rare mutations occurring during replication that isn’t caught by DNA polymerase proofreading • For mutations occurring with daily assault • If no repair • In germ (sex) cells  inherited diseases • In somatic (regular) cells  cancer

16. Protein Synthesis Transcription and Translation Mader Biology Chapter 14

17. Protein synthesis = Central dogma= DNA To RNA TO protein

18. DNA TO RNA TO PROTEIN • Transcription: DNA transcribes code to mRNA in nucleus • Translation: tRNA transports amino acids to mRNA on ribosome • Protein Synthesis-assembly of amino acids to polypeptides and, ultimately, proteins

19. 3 rna’s used to make proteins • mRNA – carries the “message” of DNA • Travels from nucleus to cytoplasm • Takes message to ribosome for protein synthesis • tRNA – brings amino acids to ribosome for protein synthesis • rRNA – what ribosome is made of

20. Amino acid attachment site A A G 3 5 Anticodon Symbol used in some books The tRNA molecule

21. Nuclear envelope DNA TRANSCRIPTION DNA TRANSCRIPTION mRNA Ribosome Pre-mRNA RNA PROCESSING TRANSLATION mRNA Polypeptide Ribosome (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. TRANSLATION Polypeptide (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Transcription and translation • In eukaryotes, separated by nuclear envelope • In prokaryotes, occurs together

22. transcription DNA mRNA

23. Non-template strand of DNA Elongation RNA nucleotides RNA polymerase T A C C A T A T C 3 U 3 end T G A U G G A G E A C C C A 5 A A T A G G T T Template strand of DNA 5 Direction of transcription (“downstream”) Newly made RNA Transcription • Transfer of genetic information from DNA to RNA (mRNA) • Similar to replication: • The DNA strands must separate. • Carried out by RNA polymerase (not DNA polymerase) • Other similar enzymes • Unlike replication: • Only 1 mRNA strand created, not 2 • 3 stages: • Initiation • Elongation • Termination

25. More Transcription • Promoter- region where RNA polymerase attaches and initiates transcription • Terminator- DNA sequence that signals the end of transcription

26. transcription DNA mRNA

27. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C Codons 5 3 mRNA Translation • mRNA  protein • Takes place on ribosome in cytoplasm

28. Translation: Genetic Information is “read” • DNA = letters A, C, T, G (you know this!) • These bases are encoded as a sequence base triplets, or codons, each of which is translated into a specific amino acid • FOR EXAMPLE: • Theredfoxatetherat = • the red fox ate the rat • AUGCCTUGUCGA = • AUG CCT UGU CGA (easier, huh?)

29. Translation: Genetic information is “read” • Codons = genetic code • Translates to amino acids (proteins) • Codon chart is universal for all living organisms (see pink sheet) • So, let’s take the DNA sequence: • TACGGT • mRNA sequence? • Amino acid sequence? • *see chart

30. DNA TRANSCRIPTION RNA is transcribed from a DNA template. 1 4 3 2 5 3 Poly-A RNA transcript RNA polymerase 5 Exon RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Cap NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM After leaving the nucleus, mRNA attaches to the ribosome. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide mRNA Activated amino acid Poly-A Poly-A Ribosomal subunits Cap 5 TRANSLATION C C A U A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) A E A C Anticodon A A A U G U G G U U U A Codon Ribosome summary

32. Protein Synthesis and Mutation • Mutation = Permanent, (sometimes) heritable DNA change • Point mutation (base substitutions) • Missense mutation • Nonsense mutation (premature stop) • Silent mutation • Frameshift mutations • Insertions or deletions • Dramatic change in amino acids • Run-ons, premature stops (nonsense mut.)

33. Protein synthesis and mutation • The Creation of Mutation (mutagenesis): • Spontaneous mutation • Occurs in DNA replication (1 in 109 bp) • Polymerase makes mistakes • Chemical mutagens (alcohol, tobacco, drugs) • Increases rate to 1 in 1000-100,000) • Radiation • X rays, gamma rays break DNA, bases • UV light causes knots in DNA strand

34. Mutation: Some Definitions • A heritable change in the genetic material • Mutations may be neutral, beneficial, or harmful • Mutagen: Agent that causes mutations • Spontaneous mutations: Occur in the absence of a mutagen

35. Substitutions • ADDING a nucleotide

36. Insertion or deletion • Called a “frameshift mutation” – affects CODONS THEBIGCATATETHERAT = THE BIG CAT ATE THE RAT THEBIGCBATATETHERAT = THE BIG CBA TAT ETH ERA T

37. Summary: Mutations Substitution mutations-one base pair is changed FRAMESHIFT: • Insertions (additions) • Deletions • Mutagens-physical and chemical agents that interact DNA to cause mutations—exp.: xrays