The Molecular Basis

of Inheritance

1
Chapter 13. Molecular Basis of Inheritance

13.1: DNA is the genetic material

13.2: Many proteins work together in DNA replication and repair

13.3: A chromosome consists of a DNA molecule packed together

with proteins

13.4: Understanding DNA structure & replication makes

genetic engineering possible

2
Overview: Life’s Operating Instructions
 In 1953, James Watson and Francis Crick introduced an elegant double-helical
model for the structure of DNA (deoxyribonucleic acid)

 How is DNA copied in cells during DNA replication & how are any errors
corrected during DNA repair?

3
 13.1: DNA is the genetic material

 Genes are located on chromosomes

 The 2 components of chromosomes—DNA & protein

Q) What is the genetic material? DNA or protein ?

4
 DNA is the genetic material

 In 1952, Hershey and Chase addressed

this by infecting viruses, called T2 phages
(35S) into bacteria
Protein : T2 phage (a kind of bacteriophages)
coat
Bacteriophages
(T2 phages)

DNA (32P)  A virus is made up of DNA (or RNA)

enclosed by a protective protein coat
Tail
: Viruses infect, enter cells and take over the host cells’
sheath
metabolic machinery in order to reproduce
Tail fiber
Genetic
Q) Is DNA or proteins the genetic material?
material
100 nm

Bacterial  Experiments: tag and trace radioactive

cell sulfur (35S) for protein
Figure 13.4 phosphorus (32P) for DNA

Conclusion: only the DNA of the T2 phage, not the protein,

enters bacteria during infection
5
Figure 13.5
Experiment
Batch 1: Radioactive sulfur (35S) in phage protein
Centrifuged cells form
Labeled phages Agitation frees outside a pellet. Free phages and phage
infect cells. phage parts from cells. parts remain in liquid.
Radioactive
Radioactivity
Protein- 35S (phage protein)
found in liquid

DNA Protein- 35S

(outside)

Centrifuge
Pellet
Batch 2: Radioactive phosphorus (32P) in phage DNA
Radioactive

DNA- 32P

DNA- 32P!
(inside)

Centrifuge
Radioactivity (phage
Pellet DNA) found in pellet
6
Animation: Hershey-Chase Experiment

7
Animation: Phage T2 Reproduction

8
DNA Is the Genetic Material

Figure 13.6
 Basic unit of DNA
1) a sugar
2) a phosphate group
3) a nitrogenous Phosphate
base

3 end
Sugar
DNA (deoxyribose)
nucleotide Nitrogenous
base

DNA: DeoxyriboNucleic Acid

9
 Deoxyribonucleic acid Ribonucleic acid

"deoxy“ means
lacking one oxygen atom at C2

10
DNA Is the Genetic Material

Figure 13.6
 Basic unit of DNA
1) a sugar
2) a phosphate group
3) a nitrogenous Phosphate
base

3 end
Sugar
DNA (deoxyribose)
nucleotide Nitrogenous
base

DNA: DeoxyriboNucleic Acid

11
DNA Is the Genetic Material
Phosphate Bases
: hydrophilic : hydrophobic

Sugar 5 end Nitrogenous bases

 Basic unit of DNA phosphate
1) a sugar backbone Thymine (T)
2) a phosphate group 5’
3) a nitrogenous
base Adenine (A)

 DNA is a polymer of Cytosine (C)

nucleotides, each
consisting of those 3
above.
Guanine (G)
3’
DNA 3 end
nucleotide
Figure 13.6
12
Building a Structural Model of DNA
 James Watson & Francis Crick first to determined the
structure of DNA
C G
: hinted by Franklin’s X-ray crystallographic images C G
G C
G C
DNA molecule is made up of two strands,
3.4 nm
forming a DNA double helix T A
G C
C G
A T

1 nm
T A
C G
G C
C G

A T
A T
Rosalind Franklin X-ray diffraction 0.34 nm
T A
photograph of DNA

13
DNA Double Helix Model Sugar-phosphate
backbone

C G
 Right handed 2 strands, C G

 antiparallel double helix 5’ G C 3’

G C
: 2 strands runs in opposite directions
3.4 nm
T A
 Sugar-phosphate backbone is outside G C
: hydrophilic phosphate group facing outside C G

 Bases (hydrophobic bases inside) A T

: purine-pyrimidine base pairing 1 nm

T A
(constant width of double helix)
C G
G C
 AT or GC base pairing
3’ C G
5’
 0.34 nm between each base pairs
 10 bases making 3.4 nm/one full turn A T
A T
0.34 nm
T A

14
 Purine-Pyrimidine base pairing for uniform width of double helix,
consistent with X-ray data

Purines: 2-carbon nitrogen ring bases (A,G)

<Purine> <Pyrimidine>
Pyrimidine: 1-carbon nitrogen ring bases (T, C) 2-carbon ring 1-carbon ring

Purine  purine

: too wide

Pyrimidine  pyrimidine

: too narrow
Adenine (A) Thymine (T)

Figure 13.9

Guanine (G) Cytosine (C)

15
  Right handed ANTIPARALLEL double helix
 Sugar-phosphate backbone (outside)
 Base (inside)
: purine-pyrimidine base pairing
Figure 13.8
5 end
C G
C G Hydrogen bond
3 end
G C
G C T A

3.4 nm
T A
G C G C
C G
A T

1 nm C G
T A
C G
G C
C G A T

A T 3 end
A T
0.34 nm 5 end
T A

(a) DNA structure (b) Partial chemical structure (c) Space-filling model
16
Animation: DNA and RNA Structure

17
Animation: DNA Double Helix

18
 13.2: DNA replication and repair

Important concepts

1) DNA replication: semiconservative model

2) Replication origin, replication fork
3) Replication machine: large protein complex
4) Antiparallel, DNA polymerase can elongate only from 5’ to 3’
o Leading strand elongation (continuous)
o Lagging strand elongation (short fragments)

19
 DNA replication: semiconservative model

templates templates

5 3 5 3 5 3 5 3
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
3 5 3 5 3 5 3 5

(a) Parental (b) Separation of parental (c) Formation of new strands

molecule strands into templates complementary to template strands

Figure 13.11-s3

 Two strands of DNA are complementary

 First step is the separation of parental strands into single strands
 Each parental strand acts as a template for building a new strand in replication
20
DNA Replication
 Remarkable accuracy
 Enzymes and other proteins involved
 Similar processes between prokaryotes (bacteria) and eukaryotes

 Origins of replication: sites where replication begins, where the two DNA strands
are separated, opening up a replication “bubble”

 Replication fork: sites at each end of a bubble, a Y-shaped region where the
parental strands of DNA are being unwound

origins of replication

replication fork

21
Figure 13.15

Origin of DNA replication

E. Coli Eukaryotic Cell

Chromosome circular linear

No. of Replication origin 1 origin many origins

1 bubble expands many bubbles are fused

E. coli Eukaryotic Cell

Parental (template) strand Origin of Double-stranded

Origin of replication DNA molecule
replication Daughter (new)
strand
Parental Daughter
Replication (template) strand (new) strand
Double- fork
stranded
DNA Replication
molecule bubble
Bubble Replication fork
Two daughter
DNA molecules

Two daughter DNA molecules

22
Animation: Origins of Replication

23
 13.2: DNA replication and repair
1) DNA replication: semiconservative model
2) Replication origin, replication fork
3) Replication machine: large protein complex
4) Due to 2 features
o DNA is antiparallel,
o DNA polymerase can elongate only from 5’ to 3’ direction
new strand formation occurs in 2 different ways (next slide)
 Leading strand: continuous elongation
 Lagging strand: production of short fragments, then joining of short fragments
New strand Template strand 5
5 3 5 3

Sugar A T A T
Phosphate Base
3
C G 3 C G

DNA
G C poly- G C
merase
3 A T A

P Pi
C 3 C 5
Pyro-
phosphate
Nucleotide 5 2 Pi 5

3 phosphate groups + a sugar (deoxyribose) + a base 24

DNA Replication: Leading vs. Lagging strand synthesis
 DNA polymerase can elongate only from 5’ to 3’
Origin of replication
Lagging
strand 3
Leading Lagging
strand strand
 Antiparallel 5 3
5 3
double helix 3 5 3 5
Leading
strand
5
Overall directions of replication

New strand elongation Leading strand Lagging strand

DNA polymerase works Toward replication fork Away from replication fork

Continuous elongation Short Okazaki fragments

Proteins/enzymes Common) Helicase, SSB, primase, RNA primer, Topoisomerase,

DNA polymerase III

---- DNA polymerase I

---- DNA ligase

25
Animation: DNA Replication Overview

26
DNA Replication: Leading vs. Lagging strand synthesis
DNA polymerase can elongate only from 5’ to 3’
Origin of replication

3
Leading
strand
5 3
5 3
3 5
Leading
strand
5
Overall directions of replication

Leading strand Lagging strand

DNA polymerase works Toward replication fork Away from replication fork

Continuous elongation Short Okazaki fragments

Proteins/enzymes Common) Helicase, SSB, primase, Topoisomerase,

Primase, RNA primer, DNA polymerase III

---- DNA polymerase I

---- DNA ligase

27
Initiation of DNA Replication
1. Helicases: enzymes that untwist the double helix at the replication forks
2. Single-strand binding proteins: proteins that bind to and stabilize single-stranded DNA
3. Topoisomerase: enzyme that relieves the strain caused by tight twisting
ahead of the replication fork by breaking, swiveling, and rejoining DNA strands

Primase

Topoisomerase
3
RNA primer
5
3
5
Replication fork
3

5
Helicase
Figure 13.14
Single-strand (ss) binding proteins

28
Initiation of DNA Replication
4. Primase: enzyme that synthesizes RNA primers, using the parental DNA as a template

5. RNA primer: initial short (5-10 nucleotide long) nucleotide chain synthesized by Primases
o DNA polymerases cannot initiate synthesis of a polynucleotide

6. DNA polymerase III: enzyme that synthesize new DNA strand from the 3’end of the RNA primer

Primase

Topoisomerase
3
RNA primer
5
3
5
Replication fork
3

5
Helicase
Figure 13.14
Single-strand (ss) binding proteins

29
1) Leading strand synthesis

DNA polymerase III (DNA pol III)

: enzyme to catalyze the synthesis of new DNA by adding
nucleotides at the 3’ end of a preexisting chain Sliding clamp: move DNA pol III along
the DNA template strand like a doughnut

Topoisomerase 3

RNA primer
5
Replication fork
3
Helicase
Single-strand (ss) binding proteins
DNA pol III: continuous elongation of a
leading strand (5’ to 3’) 3

5

3
Figure 13.17

30
Figure 13.19

Origin of replication

Leading strand Lagging

Leading strand strand
template
Single-strand 3
Leading
binding proteins Lagging strand strand
5
Leading strand
Helicase
3 5
5 DNA pol III Continuous synthesis of leading strand
3 Primer
3 5 3 Primase Lagging strand
Parental DNA DNA pol III
5 DNA pol I
Lagging strand 3 DNA ligase
template 5 5 3
3
2 5
1
Lagging strand elongate as short fragments
that later get connected

31
2) Lagging strand synthesis

Origin of replication

Leading Lagging
strand strand

Leading
Lagging
strand
strand

Directions of replication

 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

7. DNA polymerase I (cf. DNA pol III)

: enzyme to remove the RNA primers and replaces it by DNA nucleotides
8. DNA ligase: enzyme to join Okazaki fragments/ remaining gaps

32
Figure 13.18
Origin of replication
Lagging strand synthesis
Leading Lagging
strand strand

Lagging Leading RNA primer

strand strand for fragment 2
directions
of replication 5 Okazaki
DNA pol III
3 Primase makes Primer for
3 fragment 2
makes Okazaki
RNA primer. leading fragment 2.
strand
5 3 3
Template 5 3 5
strand 5
5
DNA pol III 3 DNA pol I
3 RNA primer makes Okazaki replaces RNA
for fragment 1 fragment 1. with DNA.
3
5 3 5 3 5
5 DNA ligase forms
bonds between
DNA pol III 5
DNA fragments.
detaches. 3
3
5 Okazaki

5
3 fragment 1 3
3 5
5 Overall direction of replication
33
Figure 13.18

Lagging Lagging strand synthesis

Leading strand
Lagging
strand strand

Leading RNA primer

strand for fragment 2
5
DNA pol III
3 Primase makes Primer for
3
makes Okazaki
RNA primer. Origin of leading fragment 2.
replication strand
5 3 3
Template 5 3 5
strand 5
5
DNA pol III 3 DNA pol I
3 RNA primer makes Okazaki replaces RNA
for fragment 1 fragment 1. with DNA.
3
5 3 5 3 5
5 DNA ligase forms
bonds between
DNA pol III 5
DNA fragments.
detaches. 3
3
5
5
3 3
3 5
5 Overall direction of replication
34
Okazaki Fragment1
Figure 13.18
Origin of replication
Lagging Lagging strand synthesis
Leading strand
Lagging
strand strand

Leading RNA primer

strand for fragment 2
5
DNA pol III
3 Primase makes Primer for
3
makes Okazaki
RNA primer. Origin of leading fragment 2.
replication strand
5 3 3
Template 5 3 5
strand 5
Okazaki
5 fragment 2
DNA pol III 3 DNA pol I
3 RNA primer makes Okazaki replaces RNA
for fragment 1 fragment 1. with DNA.
3
5 3 5 3 5
5

DNA pol III 5 DNA ligase

detaches. 3 Link DNA fragments.
3
5 Okazaki

5
3 fragment 1 3
3 5
5
Overall direction of replication
35
DNA Replication: Leading vs. Lagging strand synthesis
DNA polymerase can elongate only from 5’ to 3’
Origin of replication
Lagging
strand
Leading Lagging
strand strand
5 3
5 3
5 3
3
3 5 3 5 5
Leading
strand

Overall directions of replication

Leading strand Lagging strand

DNA polymerase works Toward replication fork Away from replication fork

Continuous elongation Short Okazaki fragments

Proteins/enzymes Common) Helicase, SSB, primase, Topoisomerase,

Primase, RNA primer, DNA polymerase III

---- DNA polymerase I

---- DNA ligase

36
Animation: Leading Strand

37
Animation: Lagging Strand

38
The DNA Replication Complex: simultaneous leading & lagging strand synthesis
 The DNA replication machine may be stationary during the replication process
 Recent studies support a model: 2 DNA polymerase molecules “reel in” parental
DNA and “extrude” newly made daughter DNA molecules

Leading strand template

DNA pol III

Leading strand
Parental DNA 5
5 3 3

3 5 3
5
Connecting protein
DNA pol III Helicase

Lagging 3 5
strand 3 Lagging strand
5
template

Figure 13.20
Overall direction of replication

39
Animation: DNA Replication

40
 Proofreading and Repairing DNA  Nucleotide excision repair:
: complimentary base pairing to templates
1) Nuclease: cuts out damaged
stretches of DNA
2) DNA polymerase: replaces it by
Q1) Replication errors during DNA replication? correct one
 DNA polymerases 3) DNA ligase: fill gaps
: proofread each nucleotide (nt) against its template as soon
as it is added to the growing strand, if not, replace Thymidine dimers
incorrect nt.
 Mismatch repair enzymes 5 3

: detect incorrectly paired nts and corrects DNA base pairing 3 5

errors Nuclease

5 3

Q2) DNA damage by exposure to harmful 3 5

chemical or physical agents? DNA
polymerase
e.g. cigarette smoke and X-rays
 >100 repair enzymes: fix damaged DNA nts 5 3
3 5

DNA ligase
Defects in DNA repair lead to accumulation of DNA base pairing
errors in some cancers 5 3
3 5

41
 Evolutionary Significance of Altered DNA Nucleotides

 The error rate after proofreading repair is low but not zero
 Sequence changes may become permanent and can be passed on to the next
generation
 Mutations (permanent changes in the DNA sequence) are the source of the
genetic variation upon which natural selection operates

42
Replicating the Ends of DNA Molecules
 Telomere
: special nucleotide sequences present in eukaryotic chromosomal DNA at their ends.
: repetitive sequence that does not include genes (It acts as a buffer zone to protect genes)
: For linear DNA, the usual replication machinery cannot complete the 5 ends of daughter
strands, producing shorter DNA molecules after repeated replication

 Telomerase: enzyme that catalyze the lengthening of telomeres, protecting

against telomere shortening (in germ cells)

Telomere

Figure 13.22 Telomere

Telomerase

1 m

Telomeres shorten as cell divide

43
 13.3: A chromosome consists of a DNA molecule
packed together with proteins

Q) How DNA (2 meter long) is packaged into chromosomes?

6um

1XFOHRLG

Bacteria (Prokaryotes) Eukaryotes

1-2 circular, supercoiled DNA Multiple linear DNA

DNA + proteins exist as a dense region, DNA + proteins (histones) inside of nuclear
called nucleoid (no membrane) membrane

No separate nucleus Nucleus surrounded by membrane

44
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

45
 A nucleosome
consists of
DNA wound
twice around a
protein core of
8 histones 4. Looped domain
(300-nm fiber)
Chromatid
(700 nm)
2. Nucleosome
(10 nm in diameter)
1. DNA
double helix
(2 nm in diameter) Looped
domain Scaffold
H1 300-nm
Histone tail
fiber
Histones 3. 30-nm fiber
Histones are proteins,
responsible for the first
level of DNA packing in 5. Metaphase
chromatin
chromosome
: 4 types of histones are
most common in chromatin (1,400 nm)
(H2A, H2B, H3, and H4)

Figure 13.23 (Page 312-313) 46

 A nucleosome
consists of
DNA wound
twice around a
protein core of
8 histones 4. Looped domain
(300-nm fiber)
Chromatid
(700 nm)
2. Nucleosome
(10 nm in diameter)
1. DNA
double helix
(2 nm in diameter) Looped
domain Scaffold
H1 300-nm
Histone tail
fiber
Histones 3. 30-nm fiber
Histones are responsible : Histone H1-mediated
for the first level of DNA interaction between
packing in chromatin the histone tails of 5. Metaphase
: 4 types of histones are nucleosomes and the chromosome
most common in chromatin linker DNA
(H2A, H2B, H3, and H4) (1,400 nm)
: 10nm fiber coils to
form 30 nm fiber
Figure 13.23 (Page 312-313) 47
 A nucleosome Looped domains
consists of attached to a
DNA wound chromosome scaffold,
twice around a forming 300nm fiber
protein core of
8 histones 4. Looped domain
(300-nm fiber)
Chromatid
(700 nm)
2. Nucleosome
(10 nm in diameter)
1. DNA
double helix
(2 nm in diameter) Looped
domain Scaffold
H1 300-nm
Histone tail
fiber
Histones 3. 30-nm fiber
Histones are responsible : Histone H1-mediated
for the first level of DNA interaction between
packing in chromatin the histone tails of 5. Metaphase
: 4 types of histones are nucleosomes and the chromosome
most common in chromatin linker DNA
(H2A, H2B, H3, and H4) (1,400 nm)
: 10nm fiber coils to
form 30 nm fiber
Figure 13.23 (Page 312-313) 48
The Degree of DNA Packing Affects Transcription
; a condensed region may be loosened or modified as needed for various cell processes
e.g. histones or DNA modifications changes chromatin organization
Heterochromatin
 Heterochromatin Telomere

: more condensed chromatin, transcriptionally less active

(largely inaccessible to transcription machinery)
 Euchromatin Centromere
: the more dispersed, less compacted chromatin,
transcriptionally more active Euchromatin

49
Animation: DNA Packing

50
 13.4: Genetic engineering
1) DNA (Gene) cloning
: used to make many copies of a gene and to produce a protein product

Figure 13.24
Bacterium Gene inserted into plasmid Cell containing gene
(a cloning vector) of interest

Bacterial Plasmid
chromosome DNA of chromosome
(“foreign” DNA)
Gene of interest
Restriction enzymes Recombinant
DNA (plasmid) Plasmid put into The same
bacterial cell Restriction enzymes
to cut both plasmid and
Recombinant foreign DNA
bacterium
Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest
Protein expressed
from gene of interest

Genetic modification Protein production

51
Animation: Restriction Enzymes

52
 Bacterial
How to Make Recombinant DNA plasmid
using Restriction Enzymes
Restriction site
5 3
DNA GA ATTC
C.T TAAG
3 5

Restriction enzyme
cuts the sugar-phosphate
backbones at each arrow.
 Bacterial restriction enzymes 5 3
G
5
3
cut DNA molecules at specific 3
5 3 5
DNA sequences called Sticky ends
restriction sites 5
3
5
DNA fragment from another 3
source is added. Base pairing
of sticky ends produces Foreign DNA Fragment
various combinations. cut by the same restriction
enzyme
 Complimentary base pairing 5 35 35 3
G AATT C G AATT C
between the sticky ends of 3
C T TAA G
53
C TTAA G
53 5
plasmid and foreign DNA, allows One possible combination
DNA ligase
to form a recombinant plasmid seals the strands.
5 3

3 Recombinant DNA molecule 5

Recombinant Figure 13.25

plasmid 53
 13.4: Genetic engineering
1) DNA (Gene) cloning
: used to make many copies of a gene and to produce a protein product

Figure 13.24
Bacterium Gene inserted into plasmid Cell containing gene
(a cloning vector) of interest

Bacterial Plasmid
chromosome DNA of chromosome
(“foreign” DNA)
Gene of interest
Recombinant
DNA (plasmid) Plasmid put into The same
bacterial cell Restriction enzyme
to cut both plasmid and
Recombinant foreign DNA
bacterium
Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest
Protein expressed
from gene of interest

Genetic modification Protein production

54
 5 3
2) PCR (Polymerase Chain Technique
Target sequence
Reaction): Amplifying DNA
Genomic DNA 3 5
in Vitro
Denaturation 5 3

3 5
Annealing
Cycle 1
yields 2 molecules Primers

 Produce many copies of a specific Extension

target segment of DNA New
nucleotides
 Use heat-stable DNA polymerase
called Taq polymerase.

Cycle 2
yields 4 molecules

Cycle 3
2 of the 8 molecules
(in white boxes)
match target sequence Figure 13.27
and are the right length
55
1. used to amplify the specific DNA fragment to be cloned
2. PCR primers are synthesized to include a restriction site that matches the
site in the cloning vector
3. The fragment and vector are cut and ligated together
4. DNA sequencing to check at the boundary of inserted gene

Cloning DNA fragment obtained

vector by PCR (cut by same
(bacterial restriction enzyme used
plasmid) on cloning vector)
Mix and ligate

Recombinant
DNA plasmid

56
3) CRISPR-Cas9 system: Editing Genes and Genomes
Molecular scissors

Cas9 protein Guide RNA

engineered to
“guide” the Cas9
protein 2 components:
to a target gene
5
3
Complementary 1) Cas9: a nuclease that cuts double-
sequence that can stranded DNA molecules as directed by
Active sites that bind to a target gene guide RNA
can cut DNA
Cas9guide RNA complex
2) Guide RNA
o a RNA that is complementary to the
target gene
o “Guide” the Cas9 protein to a target
gene

CYTOPLASM  Cas9-Guide RNA complex

: Guide RNA brings Cas9 to specific
NUCLEUS target site of gene and cut target
Figure 13.31
sequence
57
 Figure 13.31
CYTOPLASM
CRISPR-Cas9 system Cas9 active sites NUCLEUS

Molecular scissors Guide RNA

complementary
sequence

5 3
5
3 5

a) “knock out” (disable) a given gene Part of the

target gene

: cut site were fixed with random repair, Resulting cut

in target gene
interfering the normal gene function

b) correct mutation

b) “correct a mutated gene”

Normal
(genes associated diseases) (functional)
a) knock out gene for use as
a template by
: added normal DNA as a template can correct repair enzymes
mutation, producing normal gene (a) Scientists can disable (b) If the target gene has a
(“knock out”) the target gene mutation, it can be repaired.
to study its normal function.

Random nucleotides Normal nucleotides

58
The end

59