biol 102 chp 16 powerpoint
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Chapter 16 BIOL 102
The Molecular Basis of Inheritance
Rob Swatski
Assoc. Prof. Biology
HACC – York Campus
Overview: Life’s Operating Instructions
1953: James Watson & Francis Crick
- double-helix model
- structure of deoxyribonucleic acid (DNA)
DNA directs development of traits:
- biochemical
- anatomical
- physiological
- behavioral
The Search for the Genetic Material
After Morgan’s research on genes & chromosomes, DNA & protein became likely candidates for
the genetic material
The key factor was choosing appropriate experimental organisms
The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect
them
1928: Frederick Griffith worked with 2 bacterial strains:
one pathogenic & one harmless
When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain:
some living cells became pathogenic
He referred to this as transformation
- we now define it as a change in genotype & phenotype due to assimilation of foreign DNA
Living S cells (control)
Living R cells (control)
Heat-killed S cells (control)
Mixture of heat-killed S cells & living R cells
Mouse dies Mouse dies Mouse healthy Mouse healthy
Living S cells
RESULTS
EXPERIMENT
1944: Avery, McCarty, & MacLeod announced that DNA was the transforming substance
- based on experimental evidence showing only DNA helped transform harmless bacteria into
pathogens
- many biologists remained skeptical, mainly because little was known about DNA
Bacterial cell
Phage capsid
Tail sheath
Tail fiber
DNA
10
0 n
m
More Evidence: Bacteriophages (Phages)
1952: Alfred Hershey & Barbara Chase experiments:
- showed that DNA is the genetic material of T2 phage
- Results: only 1 of the 2 components of T2 (DNA or protein) enters an E. coli cell during infection
Concluded that the phage’s injected DNA provides the genetic information
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
Centrifuge
Centrifuge
Pellet
Pellet (bacterial cells and contents)
Radioactivity (phage
protein) in liquid
Radioactivity (phage DNA)
in pellet
Additional Evidence
It was known that DNA is a polymer of nucleotides:
- nitrogenous base, a sugar, & a phosphate group
1950: Erwin Chargaff showed DNA composition varies between species
- this evidence of diversity made DNA a more credible candidate for the genetic material
Sugar–phosphate backbone
5 end
Nitrogenous bases
Thymine (T)
Adenine (A)
Cytosine (C)
Guanine (G)
DNA nucleotide
Sugar (deoxyribose) 3 end
Phosphate
Chargaff’s Rules
In any species there is an equal number of:
A and T
&
G and C
Building a Structural Model of DNA
The next challenge was to relate DNA structure with function
Maurice Wilkins & Rosalind Franklin: used X-ray crystallography to study molecular structure
- took pictures of DNA
Rosalind Franklin Franklin’s X-ray diffraction photograph of DNA
Franklin’s images of DNA enabled Watson to deduce:
- shape: double helix
- width (double-stranded)
- spacing of N-bases
(c) Space-filling model
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
Watson & Crick built double helix models to match the x-ray & chemical evidence
Franklin’s DNA structure hypothesis:
- 2 sugar-phosphate backbones
- paired nitrogenous bases in-between
Watson built a model in which the backbones were antiparallel (their subunits run in opposite
directions)
Watson & Crick first thought bases paired “like with like” (A-A, etc.)
- but this does not result in a uniform width
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Watson & Crick concluded that:
- Adenine (A) paired only with Thymine (T)
- Guanine (G) paired only with Cytosine (C)
This model explains Chargaff’s rules:
“in any organism the amount of
A = T and the amount of G = C”
Cytosine (C)
Adenine (A) Thymine (T)
Guanine (G)
DNA Replication and Repair
Watson & Crick noted that the specific base-pairing suggested a possible DNA copying mechanism
The Basic Principle: Base Pairing to a Template Strand
• Since the 2 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 & 2 new daughter strands are built based on base-pairing rules
A T
G C
T A
T A
G C
(a) Parent molecule
A T
G C
T A
T A
G C
(c) “Daughter” DNA molecules, each consisting of one parental strand & one new strand
(b) Separation of strands
A T
G C
T A
T A
G C
A T
G C
T A
T A
G C
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) & one newly made strand
Competing models:
- Conservative model: the 2 parent strands rejoin
- Dispersive model: each strand is a mix of old & new
Parent cell First
replication Second
replication
(a) Conservative model
(b) Semiconservative model
(c) Dispersive model
• Experiments by Matthew Meselson & Franklin Stahl supported the semiconservative model
• They labeled the nucleotides of the old strands with a heavy isotope of N, while any new nucleotides were labeled with a lighter isotope
EXPERIMENT
RESULTS
1
3
2
4
Bacteria cultured in medium containing 15N
Bacteria transferred to medium containing 14N
DNA sample centrifuged after 20 min (after 1st application)
DNA sample centrifuged after 20 min (after 2nd replication)
Less dense
More dense
• The 1st replication produced a band of hybrid DNA, eliminating the conservative model
• A 2nd replication produced both light & hybrid DNA, eliminating the dispersive model & supporting the semiconservative model
CONCLUSION
First replication Second replication
Conservative model
Semiconservative model
Dispersive model
• DNA replication is remarkable in its speed & accuracy
• More than a dozen enzymes & other proteins participate in DNA replication
Getting Started
• Replication begins at special sites called origins of replication, where the 2 DNA strands are separated, opening up a replication “bubble”
• A eukaryotic chromosome may have 100’s or 1000’s of origins of replication
• Replication proceeds in both directions from each origin, until the entire molecule is copied
Origin of replication Parental (template) strand
Daughter (new) strand
Replication fork
Replication bubble
Double-stranded DNA molecule
Two daughter DNA molecules
(a) Origins of replication in prokaryotes
0.5 µm
0.25 µm
Origin of replication Double-stranded DNA molecule
Parental (template) strand Daughter (new) strand
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 proteins bind to & stabilize single-stranded DNA until it can be used as a template
• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, & rejoining DNA strands
Topoisomerase
Primase
RNA primer
Helicase
Single-strand binding proteins
5
3
5
5 3
3
• DNA polymerases cannot initiate synthesis of a polynucleotide
- they can only add nucleotides to the 3 end
• The initial nucleotide strand is a short RNA primer
(5–10 nucleotides long)
• The 3 end serves as the starting point for the new DNA strand
• Primase: can start an RNA chain from scratch & adds RNA nucleotides one at a time using the parental DNA as a template
Topoisomerase
Primase
RNA primer
Helicase
Single-strand binding proteins
5
3
5
5 3
3
Synthesizing a New DNA Strand
• DNA polymerases catalyze the elongation of new DNA at a replication fork
- most require a primer & a DNA template strand
• The rate of elongation is approx. 500 nucleotides per second in bacteria & 50 per second in human cells
• Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate
• dATP supplies adenine to DNA & is similar to the ATP of energy metabolism
- the difference is in their sugars: dATP has deoxyribose while ATP has ribose
• As each dATP joins the DNA strand, it loses 2 phosphate groups as a molecule of pyrophosphate
A
C
T
G
G
G
G C
C C
C
C
A
A
A
T
T
New strand 5 end
Template strand 3 end 5 end 3 end
3 end
5 end 5 end
3 end
Base
Sugar
Phosphate
Nucleoside triphosphate
Pyrophosphate
DNA polymerase
Antiparallel Elongation
• The double helix has an antiparallel structure
- the 2 strands are oriented in opposite directions
- this affects replication
• DNA polymerases add nucleotides only to the free 3end of a growing strand
- therefore, a new DNA strand can elongate only in the 5to3direction
• Along one template strand of DNA, the DNA polymerase continuously synthesizes a leading strand, moving toward the replication fork
Leading strand
Leading strand Lagging strand
Lagging strand
Origin of replication
Primer
Overall directions of replication
Origin of replication
RNA primer
Sliding clamp
DNA pol III Parental DNA
3
5
5
5
5
5
5
3
3
3
Helicase
Single-strand binding proteins
• To elongate the other new strand (the lagging strand), DNA polymerase must work in the direction away from the replication fork
• The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase
Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions of replication
1 2
Template strand
RNA primer for fragment 1
Okazaki fragment 1
RNA primer for fragment 2
Okazaki fragment 2
Overall direction of replication
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5 5
5 5
5 5
5
2
2
2 1
1
1
1
1
The DNA Replication Complex
• The proteins that participate in DNA replication form a large complex called a “DNA replication machine”
• Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA & “extrude” newly made daughter DNA molecules
Parental DNA
DNA pol III
Leading strand
Connecting protein
Helicase
Lagging strand DNA pol III
Lagging strand template
5
5
5
5
5
5
3 3
3 3
3
3
Proofreading & Repairing DNA
• DNA polymerases proofread newly made DNA & replace any incorrect nucleotides
• DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, & certain molecules (in cigarette smoke for example); it can also undergo spontaneous changes
• Mismatch repair: repair enzymes correct errors in base pairing
• Nucleotide excision repair: a nuclease cuts out & replaces damaged stretches of DNA
Nuclease
DNA polymerase
DNA ligase
Nucleotide Excision Repair
Nuclease
Evolutionary Significance of Altered DNA Nucleotides
• Error rate after proofreading repair is low but not zero
• Sequence changes may become permanent & can be passed on to the next generation
• These changes (mutations) are the source of the genetic variation upon which natural selection operates
Replicating the Ends of DNA Molecules
• Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes
• The usual replication machinery provides no way to complete the 5 ends
- repeated rounds of replication produce shorter DNA molecules with uneven ends
Ends of parental DNA strands
Leading strand
Lagging strand
Last fragment Next-to-last fragment
Lagging strand RNA primer
Parental strand Removal of primers and replacement with DNA where a 3 end is available
Second round of replication
Further rounds of replication
New leading strand
New lagging strand
Shorter and shorter daughter molecules
3
3
3
3
3
5
5
5
5
5
Telomeres
• Nucleotide sequences at the ends of eukaryotic chromosomal DNA molecules
• Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules
- the shortening of telomeres is thought to be connected to aging
1 µm
• If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce
• 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
• The prokaryotic chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein
- the DNA is “supercoiled” & found in the nucleoid region of the cell
• Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein
• Chromatin: a complex of DNA & protein found in the nucleus of eukaryotic cells
• Chromosomes fit into the nucleus through an elaborate, multilevel system of packing
• Histones: proteins responsible for the 1st level of DNA packing in chromatin
• 10-nm fiber (diameter) – “thin” fiber
– DNA winds around histones to form strings of nucleosome “beads”
• 30-nm fiber (diameter) – “thick” fiber
– interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber
DNA double helix (2 nm diameter)
Nucleosome (10 nm “thin” fiber)
Histones Histone tail
H1
Nucleosomes, or “beads on a string” (10 nm fiber)
30 nm fiber
Chromatid (700 nm)
Loops Scaffold
300 nm fiber
Replicated chromosome (1,400 nm) 30 nm “thick”
fiber Looped domains (300 nm fiber)
Metaphase chromosome
• Most chromatin is loosely packed euchromatin in the nucleus during interphase & condenses prior to mitosis
• During interphase a few regions of chromatin (centromeres & telomeres) are highly condensed into heterochromatin
- this dense packing of chromatin makes it difficult for the cell to express genetic information coded in these regions
• Though interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus
5
m