DNA replication Chapter 16

Figure 16.1

History of DNA Griffith Mice & Strep Transformation External DNA taken in by cell

Living S cells (pathogenic control) Experiment Results Living R cells (nonpathogenic control) Heat-killed S cells (nonpathogenic control) Mouse dies Mouse healthy Mouse dies Mixture of heat- killed S cells and living R cells Living S cells

History of DNA Hershey-Chase Bacteriophages Supported heredity information was DNA

Bacteriophages

Figure 16.4 Experiment Batch 1: Radioactive sulfur ( 35 S) in phage protein Batch 2: Radioactive phosphorus ( 32 P) in phage DNA Labeled phages infect cells. Agitation frees outside phage parts from cells. Centrifuged cells form a pellet. Radioactivity (phage protein) found in liquid Pellet Centrifuge Radioactive protein Radioactive DNA Radioactivity (phage DNA) found in pellet Centrifuge Pellet

History of DNA Franklin X-ray diffraction Double helix Watson-Crick Double helix model

History of DNA Duplication Meselson and Stahl Bacteria 14 N and 15 N Semiconservative method.

Fig Parent cell First replication Second replication (a) Conservative model (b) Semiconserva- tive model (c) Dispersive model

Figure Bacteria cultured in medium with 15 N (heavy isotope) Experiment Results Conclusion Bacteria transferred to medium with 14 N (lighter isotope) DNA sample centrifuged after first replication DNA sample centrifuged after second replication Less dense More dense Predictions: First replication Second replication Conservative model Semiconservative model Dispersive model

DNA structure

Nucleic acid structure DNA deoxyribonucleic acid RNA ribonucleic acid Nucleotides

Nucleotide structure 1. 5 carbon sugar (ribose) 2. Phosphate 3. Nitrogenous base

Nucleotide structure

Nitrogenous base Purines (2 rings) Adenine(A) & Guanine(G) Pyrimidines (1 ring) Cytosine (C), Thymine (T) DNA only Uracil (U) RNA only

Phosphodiester bond Links 2 sugars (nucleotides)

Nucleic acids 5 ’ Phosphate group (5 ’ C) at one end 3 ’ Hydroxyl group (3 ’ C) at the other end Sequence of bases is expressed in the 5 ’ to 3 ’ direction GTCCAT 5 ’ pGpTpCpCpApT---OH 3 ’

Double helix Complementary Sequence on one chain of DNA Determines sequence of other chain 5 ’ -ATTGCAT-3 ’ 3 ’ -TAACGTA-5 ’

Double Helix Complementary Purines pair with pyrimidines Diameter of base pairs are the same Adenine (A) forms 2 hydrogen bonds with Thymine (T) Guanine (G) forms 3 hydrogen bonds with cytosine (C)

Double Helix Sugar-phosphates are the backbone Complementary Phosphodiester bonds Strands are antiparrellel Bases extend into interior of helix Base-pairs form to join the two strands

Fig Hydrogen bond 3 end 5 end 3.4 nm 0.34 nm 3 end 5 end (b) Partial chemical structure(a) Key features of DNA structure 1 nm

Duplication DNA unzips-breaks hydrogen bonds New strand forms based on existing strand Old strand is saved Compliment of new strand New DNA-one old strand & one new strand Semiconservative replication

Fig A T G C TA TA G C (a) Parent molecule AT GC T A T A GC (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands A T G C TA TA G C A T G C T A T A G C

Duplication Enzymes DNA helicase: Enzyme opens helix starts duplication Separates parental strands Single-strand binding protein: Binds to unpaired DNA After separation Stabilizes DNA

Duplication Enzymes DNA polymerases: Help lengthen new strand of DNA Adds new nucleotides strand Synthesis occurs only one direction 5’ to 3’ Adding new nucleotides to the 3’OH

Duplication Enzymes Primer: Section of RNA Complementary to the parental DNA Synthesis occurs only one direction 5’ to 3’ DNA primase: Enzyme creates the primer

Duplication Enzymes Topoisomerase: Relieves strain of unwinding DNA DNA pol1: Removes primers Replaces with DNA nucleotides DNA ligase: Creates phosphodiester bonds between Okazaki fragments

Table 16.1

Duplication OriC Origins of replication Starting point in DNA synthesis Replication is bidirectional Proceeds in both directions from origin 5’to 3’direction

Duplication E coli (bacteria) Circular DNA One origin Eurkaryotes Multiple origins

Origins of Replication

Duplication Replication bubble: Separation of strands of DNA Replication of DNA Replication fork: Y-shaped region End of replication bubble Site of active replication

Duplication

DNA Replication Overview

Duplication Leading strand: DNA continuous 5’ to 3’ replication (towards fork) Template is 3’ to 5’ Lagging strand: DNA duplicated in short segments (away from fork) Okazaki fragments: Short stretches of new DNA-lagging side

Duplication Unzips (helicase, single-strand binding protein, topoisomerase) Primer DNA polymerase (5’to3’) DNA ligase

Duplication

Fig A C T G G G GC CC C C A A A T T T New strand 5 end Template strand 3 end 5 end 3 end 5 end 3 end Base Sugar Phosphate Nucleoside triphosphate Pyrophosphate DNA polymerase

Leading Strand

Fig Topoisomerase Helicase Primase Single-strand binding proteins RNA primer

Fig b Origin of replication RNA primer “Sliding clamp” DNA pol III Parental DNA

Fig a Overview Origin of replication Leading strand Lagging strand Overall directions of replication 1 2

Lagging Strand

Fig Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single-strand binding protein

Fig Overview Origin of replication Leading strand Lagging strand Overall directions of replication Template strand RNA primer Okazaki fragment Overall direction of replication

Figure Leading strand template 5′5′ 5′5′ 5′5′ 5′5′ 3′3′ 3′3′ 3′3′ 3′3′ 3′3′ 3′3′ 5′5′ 5′5′ Leading strand Lagging strand Lagging strand template DNA pol III Connecting protein Helicase Parental DNA DNA pol III

Duplication

Telomers: Sequences at ends of chromosomes Short nucleotide sequences Repeated times Prevents 5’ end erosion Telomerase: Enzyme that lengthens telomers Usually in germ cells

Repairs Mismatched pair: Duplication error Enzymes remove error Nucleotide excision repair: Damaged section removed Nuclease New nucleotides fill gap Complement DNA section not damaged

Figure Nuclease 5′5′ 5′5′ 5′5′ 5′5′ 3′3′ 3′3′ 3′3′ 3′3′ 5′5′ 5′5′ 3′3′ 3′3′ DNA polymerase DNA ligase 5′5′ 3′3′5′5′ 3′3′

Chromosome packaging Chromatin: Complex composed of DNA and proteins 40% DNA 60% protein Heterochromatin: More compacted chromatin Euchromatin: Loosely packed chromatin

Figure µm

Chromosome packaging Double helix Histones: proteins Nucleosome: DNA coiled around 8 histones (10nm) Nucleosomes then coil (30nm) Looped domains attach to chromosome scaffold (300nm) Domains coil form chromosome

Figure DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome DNA double helix (2 nm in diameter) Nucleosome (10 nm in diameter) 30-nm fiber Loops Scaffold Histones Histone tail H1 300-nm fiber Chromatid (700 nm) Replicated chromosome (1,400 nm)

Figure 16.22a DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber) DNA double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histones Histone tail H1

Figure 16.22b 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome 30-nm fiber Loops Scaffold 300-nm fiber Chromatid (700 nm) Replicated chromosome (1,400 nm)

DNA Packing