Download Chapter 11

DNA Replication and Transcription
Biosynthesis of DNA and RNA
Replication of DNA
Action of DNA Polymerases
DNA Damage and Repair
Synthesis of RNA
Post-transcriptional Modifications of RNA
Base Sequences in DNA
1
Replication of DNA
During replication, each original strand of
DNA is used as a template.
DNA replication is semiconservative.
Each new DNA duplex is composed of an
original strand and a new one.
It has been demonstrated that this
semiconservative process is universal for
all cells.
2
DNA replication
Parent DNA
First
generation
Second
generation
semiconservative
conservative
3
Replication of DNA
Replication begins at a disecrete point on the
DNA molecule and proceeds bidirectionally.
4
DNA replication
Replication process
In the late 1950, John Cairns observed
structures during the replication of DNA
in E. coli cells - replication forks.
In circular DNA, replication is observed as
occurring at a discrete point and
proceeding in both directions.
In linear DNA, replication is initiated as
several sites which grow in both
directions - replication bubbles.
6
Circular DNA replication
replication
forks
terminus
7
Linear DNA
Replication
bubbles
Blue
Red
original
daughter
8
Action of DNA polymerases
DNA polymerase I
The first enzyme discovered that would
catalyze the synthesis of DNA.
dNTP + (dNMP)n
Mg2+
(dNMP)n+1 + PPi
dNTP
deoxyribonucleoside triphosphates
(dATP, dGTP, dCTP, dTTP)
(dNMP)
preformed DNA with n or n+1
mononucleotides
PPi
pyrophosphate
9
DNA polymerase I
• Mg2+ complexes the nucleotide.
• Energy is supplied from the release of
pyrophosphate.
• DNA acts as a template.
• The DNA must have a primer segment with
a free 3’-hydroxyl group - for attachment of
the new nucleotide.
Elongation occurs at the 3’ end and proceeds
in the 5’ -> 3’ direction.
10
DNA polymerases II and III
• Additional polymerases have been
discovered in E. coli.
• These enzymes have many of the same
reaction requirements of DNA polymerase I.
• It is now believed the DNA polymerase III is
the main enzyme used for DNA replication.
• The other forms most likely serve
proofreading or repair functions.
11
DNA polymerases
Characteristic
Molecular weight
Polypeptide subunits
Polymerization rate
(nucleotides/sec)
I
Polymerase
II
III
103,000
88,000
900,000
1
4
10
16 - 20
7
250-1000
Activity
3’ -> 5’ exonuclease
Yes
Yes
Yes
5’ -> 3’ exonuclease
Yes
No
No
12
Okazaki fragments
• All know DNA polymerases catalyze chain
elongation in the 5’ 3’ direction.
• The two template strands are oriented in an
antiparallel fashion.
• Only one strand can be processed in a
continuous fashion - 3’ 5’ parent strand.
• The complementary strand is synthesized in
the 5’ 3’ direction as discontinuous
fragments - Okazaki fragments.
13
Okazaki fragments
The fragments are still added in the 5’ 3’ direction.
They are covalently linked in later steps.
Leading strand
3’
5’
3’
5’
Lagging strand (Okazaki fragments)
14
DNA Replication
Prokaryotic DNA replication - E. coli
This multistep process involves several proteins.
Protein
Function
Helicase
Begins unwinding of DNA helix
DNA gyrase
Assists unwinding
SSB proteins
Stabilizes single DNA strands
Primase
Synthesis of RNA primer
DNA polymerase III
Elongation of chain by DNA synthesis
DNA polymerase I
Removal of RNA primer and fill in
gap with DNA
DNA ligase
Closes last phosphoester gap
15
Prokaryotic Replication
Step one
• Helicase recognizes and binds to the origin for
replication.
• It catalyzes the separation of the two DNA strands.
• DNA gyrase assists in unwinding and the
replication fork is formed.
DNA gyrase
5’
Helicase
3’
ADP
ATP
16
Prokaryotic Replication
Step two
• Exposed single strands of DNA must then be
stabilized and protected from cleavage of the
phosphodiester bonds.
• SSB proteins perform this function.
• Complementary strands are now available as
templates.
SSB protein
17
Prokaryotic Replication
Step three
• Primase initiates synthesis by producing a short
strand of RNA (4-10 nucleotides.)
• This is only required once for the leading strand.
Separate initiation is required for all Okazaki
fragments.
• DNA polymerase III can then process the 3’hydroxyl group.
DNA polymerase III
primer RNA
primase
18
Prokaryotic Replication
Step four
• After ‘priming’, DNA polymerase III can then
process the 3’-hydroxy group.
• The leading strand continues in the direction of
the advancing replication fork.
• The lagging strand fragments stop when they
reach another fragment.
19
Prokaryotic Replication
Step five
• The RNA primers are removed by the 5’->3’
nuclease action of DNA polymerase I.
• Remaining gaps are filled by DNA polymerase I.
DNA
polymerase I
20
Prokaryotic Replication
Step six
• DNA ligase is used to complete the final
phosphoester bond.
• Termination of replication occurs when the two
replication forks meet on the circular DNA.
DNA ligase
ADP
ATP
21
Eukaryotic replication
There is much we do not understand about
this more complicated process.
Important difference
Telomeres - specialized DNA ends that
consist of hundreds of repeating
hexanucleotide sequences
The sequence for humans is AGGGTT
22
Eukaryotic replication
Telomerase
• The enzyme that catalyzes the synthesis of
DNA ends.
• Unusual enzyme (ribozyme) that contains
an RNA molecule that serves as a template.
• The RNA guides the addition of the correct
nucleotides.
• Varying activity levels of telomerase may
serve to regulate cell division and aging.
23
DNA Damage and Repair
Mutation
• Sudden, random alteration of original DNA
code that changes the genotype.
•
It may be as simple as one wrong
nucleotide.
•
It can be harmful/deadly or be a positive
change. (evolution - rare)
Can be caused by chemical or environmental
factors - mutagens.
24
DNA Damage and Repair
There are ~4000 know human genetic diseases.
Many result from mutation of a single gene.
Sickle cell anemia.
Mutation of gene that makes part of hemoglobin.
Male pattern baldness.
Characteristic thinning of hair in males. Linked to a
pair of genes.
Albinism.
Lack of ability to produce tyrosinase which catalyzes
the conversion of tyrosine to DOPA.
25
Mutations
Spontaneous mutations
Changes that occur during normal genetic
and metabolic function.
Two types
• Mistakes in the incorporation of
deoxyribonucleotides during DNA
replication.
• Base modifications caused by hydrolytic
reactions.
26
Spontaneous Mutations
Final mistakes during E. coli replication are
very rare.
• 1 error in every 1010 base pairs.
• Actual error rate for base incorporation
may be much higher ( 1 in every 104-105).
• Repair mechanisms will correct most
mismatched bases.
27
Spontaneous Mutations
Replication errors are of three types:
• Point mutation - substitution of one base
pair for another.
• Insertion of one or more extra base pairs.
• Deletion of one or more base pairs.
Substitution is the most common type of
spontaneous mutation.
28
Spontaneous Mutations
E. coli cells have systems to detect and
repair mismatched bases.
The general mechanism proceeds in four steps.
• Endonuclease-catalyzed cleavage of
phosphoester bond holding the incorrect base.
• Removal of mismatched base by an exonuclease.
• Incorporation of the correct base by DNA
polymerase I or III.
• Closure of the final gap by DNA ligase.
29
Photodimerization
Exposure to UV light
can cause adjacent
thymines to
covalently link.
This results in a
distortion of the DNA
molecule and
breaks the hydrogen
bonding with the
adenine.
UV light
thymine
dimer
30
Thymine dimer repair in E. Coli
To repair the damage, a photoreactivating
enzyme binds to thymine dimer.
Visible light activates
the enzyme
which breaks
the dimer,
restoring
original structure.
The enzyme is then released
from the repaired DNA.
31
Thymine repair in humans
DNA repair is more complex than in E. Coli
requiring at least 5 enzymes.
Human repair mechanism must:
• cleave the sugar phosphate backbone
• remove bad section
• rebuild a new section
Xeroderma Pigmentosum
Genetic disorder where repair mechanism does
not work.
Can result in multiple skin cancers by age 20.
32
Induced mutation
A number of environmental factors can
induce a mutation - mutagens.
Radiation
Ionizing - X-rays,  rays, cosmic rays
Nonionizing - UV light
Intercalating agents
Flat, hydrophobic chemicals that are typically
aromatic. They can insert between stacked base
pairs resulting in insertion or deletion of bases.
33
Intercalating agents
intercalating
agent
34
Induced mutation
Chemical mutagens
• Reactant with bases - formaldehyde
• Base analogs - 2-aminopurine, 5bromouracil
• Acridine dyes - proflavin
• Alkylating agents - mustard gases
• Others - carcinogens
35
Synthesis of RNA
Some basic terms
• Template - the strand of DNA used for the
synthesis of RNA. It is read in the 3’-> 5’
direction.
• Coding strand - the ‘other’ DNA strand.
• Transcript - the RNA molecule. It is synthesized
in the 5’ -> 3’ direction.
The first base in a gene is numbered +1. Additional
bases are numbered sequentially. ‘Upstream’
bases are assigned negative numbers. There is
no zero value.
36
DNA-directed RNA synthesis
Prokaryotic cells rely on DNA-directed
polymerase (RNA polymerase)to catalyze
all steps in the transcription of RNA.
The process occurs in three stages:
Initiation
Elongation
Termination
37
RNA synthesis
38
RNA synthesis
In the first step,
RNA polymerase binds
to a promoter sequence
on the DNA chain.
This insures that
transcription occurs in
the correct direction.
The initial
reaction is to
separate the two
DNA strands.
39
RNA synthesis
initiation
sequence
termination
sequence
‘Special’ base
sequences in
the DNA strand
indicate where
RNA synthesis
starts and
stops.
40
RNA synthesis
The elongation process continues until an
entire gene is transcribed. This is
catalyzed by RNA polymerase
DNA template
NTP + (NMP)n
(NMP)n+1 + PPi
NTP
ribonucleoside triphosphate,
ATP, GTP, CTP, UTP
(NMP)
preformed RNA with n or n+1
mononucleotides
41
RNA synthesis
Once the
termination
sequence is
reached, the
new RNA
molecule
and the
RNA synthase
are released.
The DNA recoils.
42
RNA synthesis
The transcription process differs for
eukaryotic cells
• Three classes of RNA polymerases for the
transcription process (I, II, II)
• I transcribes large ribosomal RNA genes.
• II is for protein-encoding genes.
• III is used during transcription of tRNA
and 5S rRNA
43
RNA-directed RNA synthesis
An alternate mode of RNA synthesis is found
in RNA viruses.
They induce formation of RNA replicase in
the host cell and use RNA as a template.
Direction of synthesis is 5’ -> 3’.
Same basic mechanism.
RNA transcript is complementary to the RNA
template.
There are no editing, proofreading or repair
activities.
44
Post-transcriptional
modifications of RNA
Primary transcripts
Newly synthesized RNA molecules. They
are typically inactive.
Several types of post-processing may be
conducted to produce a mature form of
RNA that is active.
The processing varies based on the type
of RNA.
45
tRNA and rRNA processing
Four types of processes
• Trimming of the ends by phosphoester
bond cleavage
• Splicing to remove introns
• Addition of terminal sequences
• Hetrocyclic base removal
Prokaryotic cells do not demonstrate intron
removal.
46
tRNA post-processing
3’
- OH
A
C
C
5’
3’
5’
Several steps
some catalyzed
by ribonuclease P
pre-tRNA
tRNA
47
mRNA processing
While prokaryotic mRNA requires little or no
alteration, eukaryotic mRNA must be
modified in the nucleus before use.
There are three processes that occur
• Capping
• Poly A addition
• Splicing of coding sequences
48
Capping
Modification of the 5’ end.
• Hydrolytic removal of a phosphate from the
triphosphate functional group.
• Guanosine triphosphate (GTP) is used to attach a
GMP, resulting in a 5’ -> 5’ triphosphate covalent
linkage.
• The end guanine residue is then methylated at N7
Additional capping may include methylation
at ribose hydroxyl groups.
49
Poly A addition
Modification of the 3’ end.
Most mature mRNA have a 3’ tail of from 20
to 250 nucleotides.
• Initially an endonuclease catalyzes the
removal of a few 3’ base residues.
• Addition of adenine residues is catalyzed
by polyadenyl polymerase.
This tail is thought to stabilize mRNA by
increasing resistance to cellular nucleases.
50
Intron removal
Gene
Exon
Intron
Exon
Intron
Exon
transcription
Primary transcript
removal of introns
Splicing
Mature
RNA
reseal transcript
51
Base sequences in DNA
Efforts are underway to characterize the
entire human genome. This requires
methods for simple and inexpensive
sequencing of DNA.
Two methods meet this need.
• Maxam-Gilbert chemical cleavage
• Sanger chain-termination sequencing
52
The US human genome project
Methods for DNA sequencing are greatly
assisting this project.
It is a joint program of the Department of
Energy and the National Institutes of
Health.
This project is a part of a larger
international effort to characterize the
genomes of humans and several model
organisms.
53
22.3
22.2
22.1
p
21.2
21.3
21.1
11.4
11.3
11.23
11.22
11.21
11.1
11.1
11.2
12
X chromosome
growth control factor, X-linked
Xg blood group
ocular albinism
sensorineural deafness
anemia, sideroblastic, with
spinocerebellar ataxia
13
21.2
22.2
21.1
cleft palate
21.3
lymphoproliferative syndrome
22.1
22.3
23
24
25
q
26
27
28
Simpson dysmorphia syndrome
coagulation factor IX, hemophilia B
blue-monochromatic color blindness
coagulation factor VIIIc, hemophilia A
homosexuality, male
Y chromosome
11.3
p 11.2
11.1
11.1
11.21
11.22
11.23
ribosomal protein S4, Y-linked
testis determing factor
zinc finger protein, Y-linked
acetylserotonin methyltransferase
testis-specific protein, Y-linked
Xg blood group
q
12
stature, Y-linked