Handout for Lecture 21: Eukaryotic DNA Replication (Nov. 29, 2005)OutlineEukaryotic DNA Replication (cont.)V. Telomerase (cont.)1. Know what the “end replication problem is” and why it occurs.2. Know telomere repeats are added and how this prevents chromosome shortening.VI. Packaging of Eukaryotic DNAVII. DNA TopologyMOLECULAR BIOLOGY AND BIOCHEMISTRY 694:407 & 115:511Handout for Lecture 21: Eukaryotic DNA Replication (Nov. 29, 2005)Dr. Marty NemerofWaksman 19(732) [email protected]“I don’t care to belong to any social organization that will accept me as a member.”—Groucho Marx, in a letter declining an ofer of admission to an exclusive country club.OutlineEukaryotic DNA Replication (cont.)V. Telomerase (cont.)VI. Packaging of Eukaryotic DNAVII. DNA TopologyA. What is Supercoiling?B. Sample Questions for Calculating Linking Number C. Type I and Type II TopoisomerasesD. Mechanism of TopoisomerasesE. The Role of Topoisomerases in DNA Replication1. Relieving Positive Supercoils Caused by Strand Unwinding2. Decatenating Circular DNAs After ReplicationF. A Review of the Diferent TopoisomerasesVIII. What you need to know for Exam #4Reading ListA. Biochemistry, Garrett and Grisham1. Chapter 11 (3rd. ed.—pp. 352-358) or Chapter 12 (2nd ed.)—Structure of Nucleic Acids A. Molecular Biology of the Gene, Watson et al., 5th Edition1. Chapter 6—The Structures of DNA and RNA (pp. 111-122)2. Chapter 7—Chromosomes, Chromatin, and the Nucleosome (pp. 151-180)3. Chapter 8—The Replication of DNA (pp. 228-234)1Circular dsDNAs can be replicated completely.E. coli Eukaryotes• plasmids • mitochondrial DNA chromosome• E. coli chromosome • circular dsDNA viruses (e.g., SV40)Replication of linear dsDNAs (e.g., eukaryotic chromosomes) poses an “end replication problem.”The End Replication Problem21st Round of Replication3’--------------3’--------------3’--------------5’--------------5’--------------++3’5’3’5’2nd Round of Replication3’5’3’5’Primer gapPrimer gapPrimer gapV. Telomerase (cont.)Adding Telomeres to the 3’ Ends of Chromosomes Allows forAddition of a New Okazaki Fragment to the 5’ Ends3Key:3’--------------3’--------------3’--------------3’--------------5’--------------5’--------------5’--------------5’--------------5’--------------++5’--------------3’--------------Telomerase extends3’ ends of chromosomesAdditional 3’ end DNAcan act as a template for a new Okazaki fragmentMaturation of Okazaki fragment5’--------------3’--------------5’--------------3’--------------5’--------------3’--------------Primer gap3’5’3’3’3’5’5’Primer gapTelomere repeatsNew Okazaki fragment3’3’5’5’Adapted from Watson Fig. 8-34Parental Strands1st Round Daughter Strands2nd Round Daughter Strands3’5’5’VI. Packaging of Eukaryotic DNAA Packaging ProblemIn the human genome, there are 3 X 109 bp distributed among 23 pairs of chromosomes.There are 3.4 Å/ bp in B-DNAThe total length of the DNA in a human cell is 2 meters. But it must be packaged into a nucleus with a diameter of 5 m (in a cell with a diameter of ~20 m).Therefore, DNA must be condensed by a factor of more than 100,000To solve this packaging requirement, the DNA in a eukaryotic cell nucleus during interphase (between cell divisions) exists in a condensed form as a nucleoprotein complexcalled Chromatin.4Adapted from Watson Fig. 8-37Chromatin proteins1. Histone Proteins — small, positively charge (rich in lysine and arginine residues) — found only in eukaryotes — highly conserved evolutionarilyCore histones: H2A, H2B, H3, H4Linker histone: H12. Nonhistone chromosomal proteinsDNA Wraps Around Core Histone Octomers to Form Nucleosomes• Two molecules of each of the four core histones—H2A, H2B, H3 and H4—form a histone octomer (histone core)• DNA (core DNA) wraps around the histone cores to constitute a nucleosome.• The core DNA (146 bp) wraps around the histone core almost twice (80 bp/complete turn).• Between each nucleosome is 20-60 bp of linker DNA bound by a molecule of H1 histone.• Nucleosomes are spaced approx. 200 bp apart (146 bp core DNA + 20-60 bp linker DNA).DNA Packaged into Nucleosomes (“beads on a string”)52 H2A2 H2B2 H32 H4Histone H1 binds two DNA helices6WatsonFigure 7-18Histone H1 Induces Tighter DNA Wrapping Around the NucleosomeA Model for Chromosome Structure7WatsonFigure 7-27WatsonFigure 7-29H1 is the Linker Histone:Binds the Linker DNASolenoid= cylindrical coil8DNA exists in chromatin form during interphaseDNA is most compact in chromosome form during metaphaseof mitosisSolenoid= cylindrical coilVII. DNA Topology (the configuration of DNA)A. What is supercoiling?• The pitch of B-DNA in solution is approx. 3.4 nm/helical repeat• In addition to the helical coiling of single strands to form a double helix, the double stranded DNA molecule can also twist upon itself. This is what is known as supertwisting or “supercoiling.”Bacterial plasmids were the first molecules shown to be supercoiled. It was thought to be a special feature of only circular molecules like viral chromosomes, the E. coli chromosome and plasmids.Supercoiling occurs in nearly all chromosomes, whether circular such as bacteria or linearsuch as in eukaryotes.Relaxed DNA vs Supercoiled DNARelaxed DNA has no supercoils9G & G, 3rd ed.Fig. 11.28Negatively supercoiled DNA is underwound (favors unwinding of the double helix)(circular DNA isolated from natural sources is always negatively supercoiled)Positively supercoiled DNA is overwound (favors overwinding in the double helix)Terms for Describing DNA TopologyL = T + W1. Linking Number (L or Lk) = number of times the two strands are intertwined2. Twist (T or Tw) = number of helical turns For a 2,000 bp DNA duplex, T= 200 (2,000 bp  1 turn/10 bp = 200 turns)3. Writhe (W or Wr) = number of times the duplex crosses itself (only topologically constrained DNA molecules can have writhe)A relaxed DNA molecule has zero writhes. ( For a relaxed DNA molecule, L = T)10The Linking Number Diference (L) is the diference between the linking number of a DNA (L) and thelinking number of its relaxed form (Lo) The equation is L = L – Lo For a relaxed DNA molecule: L is 0The superhelical density ( = L/ Lo) is a measure of supercoiling that is independent of length.(For a relaxed DNA molecule:  is 0.00)DNA in cells is negatively supercoiled: