Single-strand binding protein Overall directions of replication

Single-strand binding protein Overall directions of replication
The Molecular Basis of Inheritance Chapter 16 Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Single-strand binding protein Overall directions of replication Leading strand DNA pol III 3 Primer Primase 5 3 DNA pol III Lagging strand 5 DNA pol I 4 3 5 3 2 1

Fig. 16-1 In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA Figure 16.1 How was the structure of DNA determined?

The Search for the Genetic Material: Scientific Inquiry
When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material The key factor in determining the genetic material was choosing appropriate experimental organisms The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Evidence That DNA Can Transform Bacteria
The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 Griffith worked with two strains of a bacterium, one pathogenic and one harmless heat-killed remains of the pathogenic strain + living cells of the harmless strain=living cells became pathogenic He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

EXPERIMENT RESULTS Mixture of heat-killed S cells and living R cells
Fig. 16-2 Mixture of heat-killed S cells and living R cells EXPERIMENT Living S cells (control) Living R cells (control) Heat-killed S cells (control) RESULTS Figure 16.2 Can a genetic trait be transferred between different bacterial strains? Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells

In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Phage head Tail sheath Tail fiber DNA 100 nm Bacterial cell
Fig. 16-3 Viruses that infect bacteria are called bacteriophages (or phages), are widely used in molecular genetics research Phage head Tail sheath Tail fiber Figure 16.3 Viruses infecting a bacterial cell DNA 100 nm Bacterial cell

In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig DNA was tagged with radioactive phosphorous and protein was tagged with radioactive sulfur EXPERIMENT Empty protein shell Radioactivity (phage protein) in liquid Radioactive protein Phage Bacterial cell Batch 1: radioactive sulfur (35S) DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Figure 16.4 Is protein or DNA the genetic material of phage T2? Batch 2: radioactive phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet Pellet

Additional Evidence That DNA Is the Genetic Material
It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Sugar–phosphate backbone 5 end Sugar (deoxyribose) 3 end
Fig. 16-5 Sugar–phosphate backbone  end Nitrogenous bases Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases Thymine (T) Adenine (A) Figure 16.5 The structure of a DNA strand Cytosine (C) Phosphate DNA nucleotide Sugar (deoxyribose)  end Guanine (G)

(b) Franklin’s X-ray diffraction photograph of DNA
Fig. 16-6 Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DNA molecule using this technique Figure 16.6 Rosalind Franklin and her X-ray diffraction photo of DNA (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

Fig. 16-7 Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical The width suggested that the DNA molecule was made up of two strands, forming a double helix 5 end Hydrogen bond 3 end 1 nm 3.4 nm Figure 16.7 The double helix For the Cell Biology Video Stick Model of DNA (Deoxyribonucleic Acid), go to Animation and Video Files. For the Cell Biology Video Surface Model of DNA (Deoxyribonucleic Acid), go to Animation and Video Files. 3 end 0.34 nm 5 end (a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model

Franklin had concluded that there were two antiparallel (the reading strand runs 5’-3’ the other runs opposite) sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Purine + purine: too wide
Fig. 16-UN1 purine with a pyrimidine pairing results in a uniform width consistent with the X-ray Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

Watson and Crick reasoned that the pairing was more specific, dictated by the base structures
They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Fig. 16-8
Figure 16.8 Base pairing in DNA Guanine (G) Cytosine (C)

Concept 16.2: Many proteins work together in DNA replication and repair
Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

(b) Separation of strands
Fig A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand Figure 16.9 A model for DNA replication: the basic concept

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 (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new) Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

(a) Conservative model
Fig First replication Second replication Parent cell (a) Conservative model (b) Semiconserva- tive model Figure Three alternative models of DNA replication (c) Dispersive model

Fig EXPERIMENT Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope 1 Bacteria cultured in medium containing 15N 2 Bacteria transferred to medium containing 14N RESULTS 3 DNA sample centrifuged after 20 min (after first application) 4 DNA sample centrifuged after 40 min (after second replication) Less dense More dense CONCLUSION First replication Second replication Conservative model Figure Does DNA replication follow the conservative, semiconservative, or dispersive model? Semiconservative model Dispersive model

DNA Replication: A Closer Look
The copying of DNA is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DNA replication Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Getting Started-6 main steps
1. Replication begins at special sites called origins of replication, 2. Initiation proteins bind to the Origin of Replication where the two DNA strands are separated, opening up a replication “bubble” and proceeds in both directions from each origin, until the entire molecule is copied Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig Origin of replication Parental (template) strand Daughter (new) strand Replication fork Double- stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand Figure Origins of replication in E. coli and eukaryotes 0.25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating Helicases are enzymes that untwist the double helix at the replication forks 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Single-strand binding proteins
Fig Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer Figure Some of the proteins involved in the initiation of DNA replication 5 5 3 Helicase

An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5–10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Synthesizing a New DNA Strand
3. Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Most DNA polymerases require a primer and a DNA template strand The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells 4. DNA Polymerase adds nucleotides in a 5’-3’ direction (A pairs with T, G pairs with C). Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nucleoside triphosphate
Fig New strand 5 end Template strand 3 end 5 end 3 end Sugar A T A T Base Phosphate C G C G G C G C DNA polymerase 3 end A T A Figure Incorporation of a nucleotide into a DNA strand T 3 end C Pyrophosphate C Nucleoside triphosphate 5 end 5 end

Antiparallel Elongation
5. The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication Leading strand: 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 ) toward the replication fork) 6. Lagging strand: DNA polymerase must work in the direction away from the replication fork and is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Overall directions of replication
Fig Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 DNA poll III Parental DNA Figure Synthesis of the leading strand during DNA replication 3 5 5 3 5

Fig Overview Origin of replication Leading strand Lagging strand Lagging strand 2 1 Leading strand Overall directions of replication 3 5 5 3 Template strand 3 RNA primer 3 5 1 5 Okazaki fragment 3 5 3 1 5 5 3 3 Figure 16.6 Synthesis of the lagging strand 2 1 5 3 5 3 5 2 1 5 3 3 1 5 2 Overall direction of replication

Fig a Overview Origin of replication Leading strand Lagging strand Lagging strand 2 1 Leading strand Figure 16.6 Synthesis of the lagging strand Overall directions of replication

Figure 16.6 Synthesis of the lagging strand
Fig b6 3 5 5 3 Template strand 3 5 RNA primer 3 1 5 3 Okazaki fragment 5 3 5 1 3 5 3 2 1 5 5 3 Figure 16.6 Synthesis of the lagging strand 3 5 2 1 5 3 3 1 5 2 Overall direction of replication

Table 16-1

Single-strand binding protein Overall directions of replication
Fig Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Single-strand binding protein Overall directions of replication Helicase Leading strand 5 DNA pol III 3 3 Primer Primase 5 Parental DNA 3 Figure A summary of bacterial DNA replication DNA pol III Lagging strand 5 DNA pol I DNA ligase 4 3 5 3 2 1 3 5

Proofreading and Repairing DNA
DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nuclease DNA polymerase DNA ligase Fig. 16-18
Figure Nucleotide excision repair of DNA damage DNA ligase Figure Nucleotide excision repair of DNA damage

Replicating the Ends of DNA Molecules
The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules It has been proposed that the shortening of telomeres is connected to aging Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Figure 16.19 Shortening of the ends of linear DNA molecules
5 Ends of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available Figure Shortening of the ends of linear DNA molecules 5 3 Second round of replication Figure Shortening of the ends of linear DNA molecules 5 New leading strand 3 New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules

If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Concept 16.3 A chromosome consists of a DNA molecule packed together with proteins
Bacterial chromosome double-stranded circular DNA molecule associated with a small amount of protein Eukaryotic chromosomes linear DNA molecules associated with a large amount of protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

1. DNA wrapped around proteins (histones). This is called a nucleosome
In Eukaryotic cells: Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells Levels of packaging: 1. DNA wrapped around proteins (histones). This is called a nucleosome 2. the string of nucleosome folds to form a 30nm fiber 3. Further folding= looped domains of 300nm 4. Looped domains fold= metaphase chromosome For the Cell Biology Video Cartoon and Stick Model of a Nucleosomal Particle, go to Animation and Video Files. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Nucleosomes, or “beads on a string” (10-nm fiber)
Fig a Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H1 Histone tail Histones Figure 16.21a Chromatin packing in a eukaryotic chromosome DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Looped domains (300-nm fiber) Metaphase chromosome
Fig b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber Figure 16.21b Chromatin packing in a eukaryotic chromosome Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome

Loosely packed chromatin is called euchromatin
During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Histones can undergo chemical modifications that result in changes in chromatin organization
For example, phosphorylation of a specific amino acid on a histone tail affects chromosomal behavior during meiosis Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

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