The Replication of DNA
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Mason_ch14_PPTs.pptxChapter 14 Lecture Outline
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Understanding Biology, 2nd edition
Kenneth Mason
Tod Duncan
George Johnson
Jonathan Losos
Susan Singer
Copyright © McGraw-Hill Education. Permission required for reproduction or display.
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DNA: The Genetic Material
Chapter 14
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DNA is the genetic material
Mendel and researchers who came after him unraveled the essential mystery of heredity
Hereditary traits are controlled by genes on chromosomes inherited from our parents heredity
Mendel’s work left a key question unanswered: What is a gene?
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Frederick Griffith – 1928
Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia
2 strains of Streptococcus
S strain is virulent
R strain is nonvirulent
Griffith infected mice with these strains hoping to understand the difference between the strains
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Griffith’s experiment
Griffith’s results
Live S strain cells killed the mice
Live R strain cells did not kill the mice
Heat-killed S strain cells did not kill the mice
Heat-killed S strain + live R strain cells killed the mice
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Figure 14.1 a and b
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Figure 14.1 c and d
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Transformation
Transformation
Information specifying virulence passed from the dead S strain cells into the live R strain cells
Our modern interpretation is that genetic material was actually transferred between the cells
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Avery, MacLeod, & McCarty – 1944
Repeated Griffith’s experiment using purified cell extracts
Removal of all protein from the transforming material did not destroy its ability to transform R strain cells
DNA-digesting enzymes destroyed all transforming ability
Supported DNA as the genetic material
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Hershey & Chase –1952
Investigated bacteriophages
Viruses that infect bacteria
Bacteriophage was composed of only DNA and protein
Wanted to determine which of these molecules is the genetic material that is injected into the bacteria
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Hershey & Chase
Bacteriophage DNA was labeled with radioactive phosphorus (32P)
Bacteriophage protein was labeled with radioactive sulfur (35S)
Radioactive molecules were tracked
Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage
Conclusion: DNA is the genetic material
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Figure 14.2
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DNA Structure
DNA is a nucleic acid
Composed of nucleotides
5-carbon sugar called deoxyribose
Phosphate group (PO4)
Attached to 5′ carbon of sugar
Nitrogenous base
Adenine, thymine, cytosine, guanine
Free hydroxyl group (—OH)
Attached at the 3′ carbon of sugar
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Figure 14.3
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Phosphodiester bond
Bond between adjacent nucleotides
Formed between the phosphate group of one nucleotide and the 3′ —OH of the next nucleotide
The chain of nucleotides has a 5′-to-3′ orientation
Figure 14.4
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Chargaff’s Rules
Erwin Chargaff determined that
Amount of adenine = amount of thymine
Amount of cytosine = amount of guanine
Always an equal proportion of purines (A and G) and pyrimidines (C and T)
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Rosalind Franklin
Performed X-ray diffraction studies to identify the 3-D structure
Discovered that DNA is helical
Using Maurice Wilkins’ DNA fibers, discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm
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James Watson and Francis Crick – 1953
Deduced the structure of DNA using evidence from Chargaff, Franklin, and others
Did not perform a single experiment themselves related to DNA
Proposed a double helix structure
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James Watson (left) and Francis Crick a model of DNA
Figure 14.6
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Double helix
2 strands are polymers of nucleotides
Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds
Wrap around 1 axis
Antiparallel
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Figure 14.8
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Complementarity of bases
A forms 2 hydrogen bonds with T
G forms 3 hydrogen bonds with C
Gives consistent diameter
Figure 14.9
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DNA Replication
3 possible models
Conservative model
Both strands of the parental duplex would remain intact
Semiconservative model
One strand of the parental duplex remains intact in daughter strands
Dispersive model
DNA strands would consist of mixtures of parental and newly synthesized strands
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Figure 14.10
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Figure 14.10
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Figure 14.10
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Meselson and Stahl – 1958
Bacterial cells were grown in a heavy isotope of nitrogen, 15N
All the DNA incorporated 15N
Cells were switched to media containing lighter 14N
DNA was extracted from the cells at various time intervals
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Meselson and Stahl’s Results
Conservative model = rejected
2 densities were not observed after round 1
Semiconservative model = supported
Consistent with all observations
1 band after round 1
2 bands after round 2
Dispersive model = rejected
1st round results consistent
2nd round – did not observe 1 band
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“Stahl’ s” (fix #)
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Figure 14.11
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DNA Replication
Requires 3 things
Something to copy
Parental DNA molecule
Something to do the copying
Enzymes
Building blocks to make copy
Nucleotide triphosphates
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DNA replication (2)
DNA replication includes
Initiation – replication begins
Elongation – new strands of DNA are synthesized by DNA polymerase
Termination – replication is terminated
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Figure 14.12
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DNA polymerase
Matches existing DNA bases with complementary nucleotides and links them
All have several common features
Add new bases to 3′ end of existing strands
Synthesize in 5′-to-3′ direction
Require a primer of RNA
Initiation of DNA synthesis
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Prokaryotic Replication
E. coli model
Single circular molecule of DNA
Replication begins at one origin of replication
Proceeds in both directions around the chromosome
Replicon – DNA controlled by an origin
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Figure 14.13
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DNA polymerases
E. coli has 3 DNA polymerases
DNA polymerase I (pol I)
Acts on lagging strand to remove primers and replace them with DNA
DNA polymerase II (pol II)
Involved in DNA repair processes
DNA polymerase III (pol III)
Main replication enzyme
All 3 have 3′-to-5′ exonuclease activity – proofreading
DNA pol I has 5′-to-3′ exonuclease activity
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Unwinding DNA causes torsional strain
Helicases – use energy from ATP to unwind DNA
Single-strand-binding proteins (SSBs) coat strands to keep them apart
Topoisomerase prevent supercoiling
DNA gyrase is used in replication
Figure 14.14
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Semidiscontinous
DNA polymerase can synthesize only in 1 direction
Leading strand synthesized continuously from an initial primer
Lagging strand synthesized discontinuously with multiple priming events
Okazaki fragments
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Figure 14.15
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Helicase
Partial opening of helix forms replication fork
DNA primase – RNA polymerase that makes RNA primer
RNA will be removed and replaced with DNA
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Leading-strand synthesis
Single priming event
Strand extended by DNA pol III
Processivity – subunit forms “sliding clamp” to keep it attached
Figure 14.16
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Lagging-strand synthesis
Discontinuous synthesis
DNA pol III
RNA primer made by primase for each Okazaki fragment
All RNA primers removed and replaced by DNA
DNA pol I
Backbone sealed
DNA ligase
Termination occurs at specific site
DNA gyrase unlinks 2 copies
Lagging strand
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Figure 14.17
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Replisome
Enzymes involved in DNA replication form a macromolecular assembly
2 main components
Primosome
Primase, helicase, accessory proteins
Complex of 2 DNA pol III
One for each strand
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Replication fork
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Figure 14.19 1-3
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Figure 14.19 4 and 5
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Table 14.1
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Eukaryotic Replication
Complicated by
Larger amount of DNA in multiple chromosomes
Linear structure
Basic enzymology is similar
Requires new enzymatic activity for dealing with ends only
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Origins of replication
Multiple replicons – multiple origins of replications for each chromosome
Not sequence specific; can be adjusted
Initiation phase of replication requires more factors to assemble both helicase and primase complexes onto the template, then load the polymerase with its sliding clamp unit
Primase includes both DNA and RNA polymerase
Main replication polymerase is a complex of DNA polymerase epsilon (pol ε) and DNA polymerase delta (pol δ)
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Telomeres
Specialized structures found on the ends of eukaryotic chromosomes
Protect ends of chromosomes from nucleases and maintain the integrity of linear chromosomes
Gradual shortening of chromosomes with each round of cell division
Unable to replicate last section of lagging strand
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Figure 14.21
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Telomeres
Telomeres composed of short repeated sequences of DNA
Telomerase – enzyme makes telomere section of lagging strand using an internal RNA template (not the DNA itself)
Leading strand can be replicated to the end
Telomerase developmentally regulated
Relationship between senescence and telomere length
Cancer cells generally show activation of telomerase
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Figure 14.22
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DNA Repair
Errors due to replication
DNA polymerases have proofreading ability
Mutagens – any agent that increases the number of mutations above background level
Radiation and chemicals
Importance of DNA repair is indicated by the multiplicity of repair systems that have been discovered
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DNA Repair (2)
Falls into 2 general categories
Specific repair
Targets a single kind of lesion in DNA and repairs only that damage
Nonspecific
Use a single mechanism to repair multiple kinds of lesions in DNA
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Photorepair
Specific repair mechanism
For one particular form of damage caused by UV light
Thymine dimers
Covalent link of adjacent thymine bases in DNA
Photolyase
Absorbs light in visible range
Uses this energy to cleave thymine dimer
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Figure 14.23
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Excision repair
Nonspecific repair
Damaged region is removed and replaced by DNA synthesis
3 steps
Recognition of damage
Removal of the damaged region
Resynthesis using the information on the undamaged strand as a template
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Figure 14.24
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Other repair pathways
Other forms of nonspecific repair
Error-free
DNA breakage
Error-prone
Repairs massive damage
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