DNA Replication

1
Chapter 14

• DNA Replication

2
Learning Objectives

• Diagram the process of eukaryotic vs. prokaryotic
  DNA replication
• Describe the semiconservative process of DNA
  replication
• Diagram the structure of DNA (ie what are based
  like? How are they paired, where is the sugar
  backbone located and its general overall shape)
• Name the 4 enzymes involved in DNA synthesis and
  their functions
• Assess the importance of telomeres and telomerase
• Describe the process and importance of DNA
  proofreading during replication
• List the function and components of histones

3
DNA

• Stores information in a double helix
• Structure was postulated by Watson and Crick,
  based on Xray crystallography done by Rosalind
  Franklin
• DNA molecule consists of two polynucleotide
  chains twisted around each other into a
  right-handed double helix
• Each nucleotide of the chains consists of
• Deoxyribose
• A phosphate group
• A base (adenine, thymine, guanine, or cytosine)

4
Structure

• Deoxyribose sugars are linked by phosphate groups
  to form a sugar phosphate backbone
• Two strands are held together by base pairs
• AdenineThymine, GuanineCytosine
• Each full turn of double helix is 10 base pairs

5

5' end
Phosphate
Deoxyribose (a 5-carbon sugar)
Adenine (A)
Purines (double-ring structures)
Guanine (G)
Thymine (T)
Pyrimidines (single-ring structures)
Cytosine (C)
Hydroxyl group
3' end
Fig. 14-4, p. 281
6

2 nm
5' end
3' end
Distance between each pair of bases 0.34 nm
Each full twist of the DNA double helix 3.4 nm
5-carbon sugar deoxyribose)
Nitrogenous base (guanine)
Phosphate group
Hydrogen bond
3' end
Fig. 14-6, p. 283
5' end
7
DNA replication

• DNA polymerases are the primary enzymes of DNA
  replication
• Helicases unwind DNA to expose template strands
  for new DNA synthesis
• RNA primers provide the starting point for DNA
  polymerase to begin synthesizing a new DNA chain
• One new DNA strand is synthesized continuously
  the other, discontinuously

8
Assembling Antiparallel Strands
9

• Meselson and Stahl showed that DNA replication is
  semiconservative
• Two strands of parental DNA molecule unwind
• Each is a template for the synthesis of a
  complementary copy

10

KEY
a. Semiconservative replication
Parental DNA Replicated DNA
1st replication
2nd replication
The two parental strands of DNA unwind, and each
is a template for synthesis of a new strand.
After replication has occurred, each double helix
has one old strand paired with one new strand.
Fig. 14-8a, p. 285
11
Enzymes of DNA Replication

• Helicase unwinds the DNA
• Primase synthesizes RNA primer (starting point
  for nucleotide assembly by DNA polymerases)
• DNA polymerases assemble nucleotides into a
  chain, remove primers, and fill resulting gaps
• DNA ligase closes remaining single-chain nicks

12
Telomerase

• Ends of eukaryotic chromosomes
• Short sequences repeated hundreds to thousands of
  times
• Repeats protect against chromosome shortening
  during replication
• Chromosome shortening is prevented in some cell
  types which have a telomerase enzyme (adds
  telomere repeats to chromosome ends)

13

3' end of template strand
1
3' end of DNA template unwound and ready for
replication.
Primer added and new DNA assembled from end
of primer.
2
Primer
New DNA
Primer removed.
3
Chromosome strand shortened
Gap left by primer removal
Fig. 14-13, p. 291
14

Original end of chromosome
Extra telomere repeats added by telomerase at
3 end of template strand
1
Added telomere repeats
Primer added and gap filled in
2
Primer added to chromosome end
Gap filled in
3
Primer removed original length is restored
Primer removed
Chromosome strand not shortened
Fig. 14-14, p. 291
15
DNA Synthesis

• Begins at sites that act as replication origins
• Proceeds from the origins as two replication
  forks moving in opposite directions

16

Origin
DNA double helix
Replication forks
Replication direction
Fig. 14-15, p. 292
17

Origin
Replication forks
DNA double helix
Fig. 14-19, p. 297
18
Proofreading

• If a replication error causes a base to be
  mispaired, DNA polymerase reverses and removes
  the most recently added base
• Proofreading depends on the ability of DNA
  polymerases to reverse and remove mismatched
  bases
• DNA repair corrects errors that escape
  proofreading

19

Template strand
DNA polymerase
Enzyme continues activity in the forward
direction as DNA 3 polymerase as long as the
most recently added nucleotide is correctly
paired.
1
New strand
Enzyme adds a mispaired nucleotide.
2
New strand
Enzyme reverses, acting as a deoxyribonuclease
to remove the mispaired nucleotide.
3
Enzyme resumes forward activity as a DNA
polymerase.
4
Fig. 14-16, p. 293
20

Base-pair mismatch
Template strand
Repair enzymes recognize a mispaired base and
break one chain of the DNA at the arrows.
1
New strand
The enzymes remove several to many bases,
including the mismatched base, leaving a gap in
the DNA.
2
The gap is filled in by a DNA polymerase using
the intact template strand as a guide.
3
Nick left after gap filled in
The nick left after gap filling is sealed by
DNA ligase to complete the repair.
4
Fig. 14-17, p. 293
21
DNA Organization in Eukaryotes and Prokaryotes

• Histones pack eukaryotic DNA at successive levels
  of organization
• Many nonhistone proteins have key roles in the
  regulation of gene expression
• DNA is organized more simply in prokaryotes than
  in eukaryotes

22
Chromatin

• Distributed between
• Euchromatin (loosely packed region, genes active
  in RNA transcription)
• Heterochromatin (densely packed masses, genes are
  inactive)
• Folds and packs to form thick, rodlike
  chromosomes during nuclear division

23
The Bacterial Chromosome

• Closed, circular molecule of DNA packed into
  nucleoid region of cell
• Replication begins from a single origin, proceeds
  in both directions
• Plasmids (in many bacteria) replicate
  independently of the host chromosome

24
Learning Objectives

• Diagram the process of eukaryotic vs. prokaryotic
  DNA replication
• Describe the semiconservative process of DNA
  replication
• Diagram the structure of DNA (ie what are bases
  like? How are they paired, where is the sugar
  backbone located and its general overall shape)
• Name the 4 enzymes involved in DNA synthesis and
  their functions
• Assess the importance of telomeres and telomerase
• Describe the process and importance of DNA
  proofreading during replication
• List the function of histones

25
Chapter 16 Gene regulation

• Diagram the lac operon transcription unit
• Compare and contrast the operon model of
  tryptophan and lactose metabolism
• Compare and contrast prokaryotic and eukaryotic
  gene regulation

26
Gene Expression Control

• All somatic cells in an organism are genetically
  identical
• Cells differentiate by gene expression
• Gene expression is collectively controlled
  through transcriptional regulation
• Main control Gene transcribed into mRNA
• Additional controls Posttranscriptional,
  translational and posttranslational

27
Prokaryotic Gene Expression

• Operon is the unit of transcription in
  prokaryotes
• lac operon for lactose metabolism is transcribed
  when an inducer inactivates a repressor
• Transcription of the lac operon is also
  controlled by a positive regulatory system

28
Operon Unit of Transcription

• Prokaryotic gene expression reflects life history
• Rapid, reversable response to environment
• Operon A cluster of prokaryotic genes and DNA
  sequences involved in their regulation
• RNA polymerase binds at promoter for operon
• Many genes may be transcribed into one mRNA
• Cluster of genes is transscriptional unit

29
Operon Unit of Transcription (2)

• Regulatory proteins bind at operator
• Regulatory protein coded by gene outside operon
• Repressor proteins prevent operon genes from
  being expressed
• Activator proteins turn on expression of genes
  from operon

30
lac Operon for Lactose Metabolism

• Lactose metabolism in E. Coli requires three
  genes lacZ, lacY and lacA
• lac operon contains all three genes and
  regulatory sequences
• lac operon operator sequence is between promoter
  and lacZ

31

Sequences that control the expression of the
operon
Regulatory gene
lac operon
lacI
lacA
Promoter
lacY
Operator
lacZ
DNA
Binds Lac repressor
Binds RNA polymerase
Transcription termination site
Transcription initiation site
Lac repressor
ß-Galactosidase
Permease
Transacetylase
Fig. 16-2, p. 331
32
lac Operon for Lactose Metabolism

• lac repressor stops lac operon expression
• Encoded by lacI, synthesized in active form
• Binds promoter, prevents transcription
• Allolactose made from lactose when it enters
  cell, lasts as long as lactose available
• Inducer of lac operon by binding to lac repressor
• Inducible operon because inducer increases
  expression

33

a. Lactose absent from medium
lac operon
lacI
lacZ
lacA
Promoter
Operator
lacY
DNA
Transcription blocked
When lactose is absent from the medium, the
active Lac repressor binds to the operator of the
lac operon, blocking transcription.
mRNA
RNA polymerase cannot bind to promoter
Lac repressor (active)
Fig. 16-3a, p. 332
34

b. Lactose present in medium
lac operon
lacI
lacY
lacZ
lacA
Promoter
Operator
DNA
Transcription occurs
RNA polymerase binds and transcribes operon
mRNA
mRNA
Lac repressor (active)
Translation
Inactive repressor
When lactose is present in the medium, some of it
is converted to the inducer allolactose.
Allolactose binds to the Lac repressor,
inactivating it so that it cannot bind to the
operator. This allows RNA polymerase to bind to
the promoter, and transcription of the lac operon
occurs. Translation of the mRNA produces the
three lactose metabolism enzymes.
Lactose metabolism enzymes
Binding site for inducer
Allolactose (inducer)
Fig. 16-3b, p. 332
35
Positive Regulation of lac Operon

• lac operon operates when lactose but not glucose
  is present
• Glucose more efficient energy source than lactose
• Catabolite Activator Protein (CAP) is an
  activator that stimulates gene expression
• CAP activated by cAMP
• cAMP only abundant when glucose levels are low

36

a. Lactose present glucose low or absent
CAP site
Promoter
Operator
Transcription occurs
lacI
lacZ
DNA
cAMP
CAP
mRNA
RNA polymerase
mRNA
Translation
When lactose is present and glucose is low or
absent, cAMP levels are high. cAMP binds to CAP,
activating it. Active CAP binds to the CAP site
and recruits RNA polymerase to the promoter.
Transcription then occurs.
Active CAP
Lac repressor (active)
Lactose metabolism enzymes
Allolactose (inducer)
Inactive repressor
Fig. 16-5a, p. 334
37

a. Lactose present glucose low or absent
Transcription occurs
When lactose is present and glucose is low or
absent, cAMP levels are high. cAMP binds to CAP,
activating it. Active CAP binds to the CAP site
and recruits RNA polymerase to the promoter.
Transcription then occurs.
Stepped Art
Fig. 16-5a, p. 334
38

b. Lactose present glucose present
No transcription
CAP site
Promoter Operator
lacI
lacZ
DNA
RNA polymerase binding site
Inactive CAP
RNA polymerase cannot bind
mRNA
When lactose is present and glucose is present,
cAMP levels are low. As a result, CAP is inactive
and cannot bind to the CAP site. RNA polymerase
then is unable to bind to the promoter, and no
transcription occurs.
Lac repressor (active)
Allolactose (inducer)
Inactive repressor
Fig. 16-5b, p. 334
39
Summary of lac operon

• Turn off unless Lactose is present (lac I protein
  active)
• Turn on if Lactose is present (Lac I binding to
  allolactose inactive)
• Turn on if Lactose is present (CAMP binds to CAP
  to activate
• Turn off again if Lactose AND Glucose are present
  (CAMP not available with glucose present cannot
  activate CAP)

40
Tryptophan metabolism

• No Tryp available, the cell makes it own- the
  operon is turned on
• If tryp is available, the cell does not want the
  enzymes for synthesis to be made, so the cell
  turns it off
• This is negative regulation turning off rather
  than on

41

a. Tryptophan absent from medium
RNA polymerase binds and transcribes operon
Regulatory gene
trp operon
trpR
trpD
trpC
trpB
trpA
trpE
Promoter
Operator
DNA
Transcription occurs
mRNA
Trp repressor (inactive)
mRNA
Translation
When tryptophan is absent from the medium, the
Trp repressor is inactive in binding to the
operator and transcription proceeds.
Tryptophan biosynthesis enzymes
Fig. 16-4a, p. 333
42

b. Tryptophan present in medium
trp operon
trpC
trpR
trpD
trpE
trpB
trpA
Promoter
Operator
DNA
Transcription blocked
When tryptophan is present in the medium, the
amino acid binds to, and activates, the Trp
repressor. The active repressor binds to the
operator and blocks transcription.
mRNA
RNA polymerase cannot bind to promoter
Trp repressor (inactive)
Trp repressor (active)
Tryptophan-binding site
Tryptophan (corepressor)
Fig. 16-4b, p. 333
43
Eukaryotic Transcription Regulation

• In eukaryotes, regulation of gene expression
  occurs at several levels
• Chromatin structure plays an important role in
  whether a gene is active or inactive
• Regulation of transcription initiation involves a
  genes promoter and regulatory sites
• Methylation of DNA can control gene transcription

44
Regulation of Gene Expression in Eukaryotes

• Gene expression in eukaryotes has more regulatory
  points
• Chromatin has histones
• Different types of cells
• Nuclear envelope
• Three main areas of eukaryotic regulation of gene
  expression
• Transcriptional, posttrascriptional and
  posttranslational

45
Chapter 16 Gene regulation

• Diagram the lac operon transcription unit
• Compare and contrast the operon model of
  tryptophan and lactose metabolism
• Compare and contrast prokaryotic and eukaryotic
  gene regulation