Download DNA_Replication 2015

Molecular Biology
Informational Macromolecules
DNA/RNA/PROTEINS
Cells
Chemical Machines
Coding devices
I. The Blueprint of Life:
Structure of the Bacterial
Genome
• 4.1 Macromolecules and Genes
• 4.2 The Double Helix
• 4.3 Genetic Elements: Chromosomes and
Plasmids
4.1 Macromolecules and Genes
• Functional unit of genetic information is
the gene
• Genes are in cells and are composed of
DNA
4.1 Macromolecules and Genes
• Three informational macromolecules in cell
– DNA
– RNA
– Protein
4.1 Macromolecules and Genes
• Genetic information flow can be divided into three stages
– Replication: DNA is duplicated (Figure 4.3)
– Transcription: information from DNA is transferred to RNA
• mRNA (messenger RNA): encodes polypeptides
• tRNA (transfer RNA): plays role in protein synthesis
• rRNA (ribosomal RNA): plays role in protein synthesis
– Translation: information in RNA is used to build polypeptides
Replication
Transcription
C
A
G T
T
A
DNA
Dark green strand
is template for
RNA synthesis.
C
C
G
T G
A
G
C
T T
DNA
polymerase
5′
T
A
3′
C
A C
T G
G A G
G C
G
A
T
G
G
C
G A C U
U U
C U
C
C T G A G
C
A A G G A G A G
C
C
C T
T
C
G C
G T
C A C
G
3′
G
C
5′
G
mRNA
5′
A
A
C
Protein
T G
C T
C
G
U
RNA polymerase
A
Translation
Messenger RNA is
template for
protein synthesis.
tRNA
mRNA
G
A
5′
U G C
G A C U
U
G G A G
C
C U G A
U U
G G
A G
G A C
C
C
3′
Ribosome
Figure 4.3
Microbial Genetics
Structure of INFORMATIONAL MOLECULES
STRUCTURE OF GENOME
WHY DNA?
MUTATIONS, WHY CHANGE?
PROOF DNA KEY MOLECULE
CENTRAL DOGMA
DNA MAKES RNA MAKES PROTEIN
FEATURES OF DNA
1. STORES GENETIC INFO IN ITS BASE SEQUENCES
2. GREAT PHYSICAL & CHEMICAL STABILITY
3. DNA TRANSFER GENETIC INFO TO PROGENY DNA USING
OLD STAND AS TEMPLATE FOR NEW STRAND
4. GENETIC CHANGE CAN OCCUR!! MUTATIONS, WITHOUT
LOST OF PARENT INFO
ONE DAUGHTER CELL IDENTICAL TO PARENT
ONE DAUGHTER CELL SLIGHTLY DIFFERENT
5. MUTATION: CHEMICAL ALTERATION
REPLICATION ERROR
HYDROPHOBIC !. WATER FREE KEEPS RATE LOW
2. REPAIR SYSTEM
Proof DNA Information Molecule
1.1928 Fred Griffth Streptococcus pneumoniae
TRANSFORMING PRINCIPLE
2. 1944 Oswald Avery, Colin Macloed & Maclyn
McCarty
PURIFIED Cell extract
PROVED TO BE DNA
3. Blender Experiment: Alfred Herchey & Martha Chase
E. coli and Phage T2
Transforming Principle
Streptococcus pneumoniae
Smooth colonies (S) Capsulated Virulence Factor
Rough colonies (R) Non-capsulated Non-virluent
1. Heat Killed (S) non-lethal in mice
2. Mixed Live (R) bacteria & Dead (S) Bacteria  DEATH (MICE)
3. DEAD MICE-ISOLATED (S) BACTERIA
(ALSO OCCURRED ON NUTRIENT MEDIA & CELL-FREE EXTRACT)
4. S-CELL EXTRACT = ‘TRANSFORMING PRINCIPLE” UNKNOWN!
Avery, Macloed, & Mccarty
Purified S “cell Extract” Proved to be DNA
1. Isolated DNA from cell Extract of S cells
2. Added DNA to live R cells= 1/104 was S colony
3. S cells and R cells remained after growth media
4. Polysaccharide Capsule Material + R cells- R cells
1. Chemical Test proved it was DNA
2. Physical Test proved it was DNA
3. Transforming Activity NOT LOST!
proteoases
Ribonucleases
4. Transforming Activity LOST
DNAase
Blender Experiment
E. Coli Phage T2
1.
Radiolabelled
DNA 32 P
Protein 35S
Amino Acids Cysteine/methionine
2. Mixed T2 Phage with culture of E. coli cells
3. Blended mixed after short period of ATTACHMENT
4.BLENDED MIXTURE & CENTRFUGED
5. Progeny 32 P in pellet material and 35S in supernatant
ROSALAND FRANKLIN 1951
X-RAY DIFFRACTION
DNA CRYSTALS
HELICAL STRUCTURE
CLOSELY PACKAGED/ NITROGEN SIDE CHAINS
PO4 GROUPS
EYWIN CHARGAFF 1950
BIOCHEMICAL ANALYSES
COMPLIMENARY COMPOSITION
A=T
G=C
WATSON/CRICK/WILKINS
BUILT MODEL OF DNA
DOUBLE HELIX
ANTIPARALLEL STRANDS
10 BP/HELIX TURN
EQUAL WIDTH 34A° (10-7) ANGSTRAM
MAJOR GROUVES/MINOR GROUVES
(PROTEIN BINDING)
Semiconservative
Melelson & Stahl
15N
Medium
14N 21
14N % 15N 22
4.1 Macromolecules and Genes
• Genetic information flow can be divided into three stages
– Replication: DNA is duplicated (Figure 4.3)
– Transcription: information from DNA is transferred to RNA
• mRNA (messenger RNA): encodes polypeptides
• tRNA (transfer RNA): plays role in protein synthesis
• rRNA (ribosomal RNA): plays role in protein synthesis
– Translation: information in RNA is used to build polypeptides
Polysystronic
4.1 Macromolecules and Genes
• Central dogma of molecular biology
– DNA to RNA to protein
• Eukaryotes: each gene is transcribed
individually
• Prokaryotes: multiple genes may be
transcribed together
4.3 Genetic Elements:
Chromosomes and Plasmids
• Genome: entire complement of genes in
cell or virus
• Chromosome: main genetic element in
prokaryotes
• Other genetic elements include virus
genomes, plasmids, organellar genomes,
and transposable elements
4.3 Genetic Elements:
Chromosomes and Plasmids
• Viruses contain either RNA or DNA genomes
– Can be linear or circular
– Can be single- or double-stranded
• Plasmids: replicate separately from chromosome
– Great majority are double-stranded
– Most are circular
– Generally beneficial for the cell (e.g., antibiotic
resistance)
– NOT extracellular, unlike viruses
4.3 Genetic Elements:
Chromosomes and Plasmids
• Chromosome is a genetic element with
"housekeeping" genes
– Presence of essential genes is necessary for a
genetic element to be called a chromosome
• Plasmid is a genetic element that is
expendable and rarely contains genes for
growth under all conditions
4.3 Genetic Elements:
Chromosomes and Plasmids
• Transposable elements
– Segment of DNA that can move from one site
to another site on the same or a different DNA
molecule
– Inserted into other DNA molecules
– Three main types:
• Insertion sequences
• Transposons
• Special viruses
Ways DNA GETS INTO CELLS
TRANSFORMATION: Naked DNA or Plasmids incorporated into
cell genome
Competent Host Cells
1. Chemical used to open “Porins” cell transport channels
2. Electroporation- Pulsed electrical field- electric shotgun
3. Markers used to test if cell are Transformed
Antibiotic resistantPGLO fluorscent green protein
4. Plate cells on selective growth media view colonies /UV light
Transduction/ Viruses
Need host to grow
DNA 0r RNA NOT Both
Ss or ds
Structure
Capsid coat or shell- PROTEIN/ ICOSAHEDRON
Genetic Molecule DNA or RNA
Host Specific: T2 phage/E. coli
Type of Infection
Lytic- 30 min 5000/1000 progeny cell lyses BURST
Lysogenic Phage- linear DNA incorporated cell DNA
CONJUGATION
CELL SEX
CELL TO CELL CONTACT-EXCHANGE BODY FLUIDS (DNA)
SEX PILI
F+-------F+
CONFERES RESISTANCE (ANTIBIOTIC)
DEGRADATION CAPACITY
Plasmids
4.2 The Double Helix
• Four nucelotides are found in DNA (Figure 4.1):
–
Adenine (A)
– Guanine (G)
– Cytosine (C)
– Thymine (T)
• Backbone of DNA chain is alternating phosphates and the
pentose sugar deoxyribose
• Phosphates connect 3′-carbon of one sugar to
5′-carbon of the adjacent sugar
Complementary
DNA strands
3′-Hydroxyl
5′-Phosphate
Hydrogen
bonds
Phosphodiester
bond
5′-Phosphate
3′-Hydroxyl
Figure 4.4
E coli Nucloid/Genome
Single genome
Circular
Double stranded
Complimentary binding
Anti parallel
Super coiled
Major/minor grooves
4.6 X 106 bp
2000/3000 genes
4.2 The Double Helix
• Size of DNA molecule is expressed in base
pairs
• 1,000 base pairs = 1 kilobase pair = 1 kbp
• 1 million base pairs = 1 megabase pair =
1Mbp
• E. coli genome = 4.64 Mbp
• Each base pair takes up 0.34 nm of length
along the helix
• 10 base pairs make up 1 turn of the helix
4.2 The Double Helix
• Supercoiled DNA: DNA is further twisted to save space
(Figure 4.6)
– Negative supercoiling: double helix is underwound
– Positive supercoiling: double helix is overwound
• Relaxed DNA: DNA has number of turns predicted by
number of base pairs
• Negative supercoiling is predominantly found in nature
• DNA gyrase: introduces supercoils into DNA (Figure 4.7)
Relaxed circular DNA
Nucleoid
Break one strand.
Nick
Proteins
Relaxed nicked circular DNA
Supercoiled circular DNA
Rotate one end of broken
strand around helix and seal.
Supercoiled
domain
Chromosomal DNA with supercoiled domains
Figure 4.6
DNA gyrase makes
double-strand break
One part
of circle
is laid
over the
other.
Relaxed
circle
Helix
makes
contact
in two
places.
Unbroken
helix is
passed
through
the break.
Double-strand
break resealed behind
unbroken helix
Following
DNA gyrase
activity,
two
negative
supercoils
result.
Supercoiled
DNA
Figure 4.7
DNA REPLICATION
1. Parent to offspring: Semi- conservative
2. Enormous supply of ATP to unwind strands
3. ssDNA Template using bp (sense Strand)
4. N.T. (bp) added 1 X 1 to end of growing chain
POLYMERASE ENZYME
5. Sequence of growing chain: daughter Cell
COMPLEMENTARY
to base sequence in PARENT Strand (Sense Strand)
e.g. AT
DNA Enzymes
Polymerase catalyzes addition of N.T.
synthesizes DNA 5’-------------3’
de NOVO NEEDS 3’0H for synthesis
Primase- RNA short strand (15nt)
COMPLEMENATORY TO TEMPLATE DNA
REPLICATION FORK- Bidirectional
Theta Structure
ORI- ORIGIN OF REPLICATION
LEADING STRAND- CONTINOUS (1 PRIMER)
DISCONTINOUS- PRIMED MANY TIMES
Helicases
SSB
DNA Topoisomerases
Gyrase
DNA polymerase I
DNA polymerase III
5’---3’
DNA primase
DNA ligase
REPLISOME COMPLEX
TOPOISOMERASES
T1 NICK 1 STRAND
T2 NICKS 2 STRANDS
GYRASE- REMOVES SUPERCOILING
HELICASE-
UNWINDS DNA STRANDS
PRIMASE- SYNTHESIS OF RNA PRIMER
SSB- SINGLE STRANDED DNA BINDING
PROTEINS
Figure 4.9
4.3 Genetic Elements:
Chromosomes and Plasmids
• The Escherichia coli chromosome
• Escherichia coli is a useful model organism
for the study of biochemistry, genetics, and
bacterial physiology
• The E. coli chromosome from strain
MG1655 has been mapped using
conjugation, transduction, molecular
cloning, and sequencing (Figure 4.8)
Figure 4.8
4.3 Genetic Elements:
Chromosomes and Plasmids
• Some features of the E. coli chromosome
– Many genes encoding enzymes of a single
biochemical pathway are clustered into operons
– Operons are equally distributed on both strands
– ~5 Mbp in size
– ~40% of predicted proteins are of unknown
function
– Average protein contains ~300 amino acids
– Insertion sequences (IS elements)
II. Transmission of Genetic
Information: DNA Replication
• 4.4 Templates and Enzymes
• 4.5 The Replication Fork
• 4.6 Bidirectional Replication and the
Replisome
5′
3′
5′
5′
3′
3′
Parental
strand
Semiconservative
replication
+
Daughter
strand
Figure 4.11
Growing
point
DNA polymerase
activity. PPi
cleaved off
Deoxyribonucleoside
triphosphate
Figure 4.12
4.4 Templates and Enzymes
• DNA polymerases catalyze the addition of
dNTPs
• Five different DNA polymerases in E. coli
– DNA polymerase III is primary enzyme
replicating chromosomal DNA
• DNA polymerases require a primer (Figure
4.13)
– Primer made from RNA by primase
RNA primer
5′
DNA
3′–OH
G UC U U A C U G A T C A GG T T C A T C GG A CG T A T C
C AG AA T G A C T AG T CC AA G T AG C C T G C A T A G AG CC T T A CG A T C AGG C A G T
3′
5′
Figure 4.13
4.5 The Replication Fork
• DNA synthesis begins at the origin of replication
in prokaryotes
• Replication fork: zone of unwound DNA where
replication occurs (Figure 4.14)
• DNA helicase unwinds the DNA
• Extension of DNA (Figure 4.15)
– Occurs continuously on the leading strand
– Discontinuously on the lagging strand
• Okazaki fragments are on lagging strand
3′
Replication fork
Helicase
5′
ATP
3′
ADP + Pi
Helicase direction
5′
Figure 4.14
3′
5′
RNA primer
Single-strand
binding protein
5′
Leading strand
DNA polymerase III
3′
Lagging strand
Helicase
Primase
RNA primer
5′
3′
Figure 4.15
4.5 The Replication Fork
• Connecting DNA fragments on the lagging
strand (Figure 4.16)
– DNA synthesis on lagging strand continues
until it reaches previously synthesized DNA
– DNA polymerase I removes the RNA primer
and replaces it with DNA
– DNA ligase seals nicks in the DNA
Origin of
replication
Replication
forks
Newly
synthesized
DNA
Theta structure
Movement
3′
Origin
(DnaA binding site)
5′ Lagging 3′
Leading
3′
5′
5′
Replication
fork
Movement
5′
5′ Lagging 3′
Leading
3′
5′
Origin
3′
Replication
fork
Figure 4.17
5′
3′
DNA polymerase III
3′-OH
RNA primer
3′
5′
5′-P
5′
3′
3′
5′
DNA polymerase I
5′
3′
Excised RNA
primer
3′
5′
DNA ligase
3′-OH 5′-P
5′
3′
3′
5′
5′
3′
3′
5′
Figure 4.16
Helicase
Direction of
new synthesis
Newly synthesized strand
DNA polymerase III
5′
3′
RNA primer
Leading strand template
DNA helicase
DNA gyrase
5′
3′
Tau
Parental DNA
RNA primer
DNA polymerase III
3′
DNA primase
5′
5′
5′
Lagging
strand
template
Newly synthesized strand
Direction of
new synthesis
Single-strand
DNA-binding
proteins
Figure 4.18
4.6 Bidirectional Replication and
the Replisome
• DNA replication is extremely accurate
– Proofreading helps to ensure high fidelity
• Mutation rates in cells are 10–8 to 10–11 errors per
base inserted
• Polymerase can detect mismatch through
incorrect hydrogen bonding
• Proofreading occurs in prokaryotes, eukaryotes,
and viral DNA replication systems
Termination
DETAILS NOT KNOWN
Link as a CHAIN
UNLINKED BY TOPOISOMERASES
Coupled with cell wall synthesis
DNA PARTITIONED EQUALLY INTO DAUGHTER
CELLS
CELL DIVISION PROTEIN “ORCHESTRATES”
FtsZ
POLYMERIZATION
Precursors dATP, dCTP, dGTP, dTTP
Ribonucleotide Reductase
Polymerase : DNA Kinase 5’---------3’
Ori Theta formation
Primosomes- Primase, and Polymerases
Helicase Seperates DNA Strands
Gyrase- unwinds DNA
Topoisomerase I & II
Ligase – Reseals DNA stands