Transcript Slide 1
Bio3124
Lecture 8
DNA Contains Cell Information
• Total cell DNA = genome
(chromosome & extra-chromosomal)
• Human genome = 4 billion bp
– 1000x as large as E. coli genome
– 90% junk DNA
– ~8x more genes: 30,000 (human)
vs. 4,000 (E. coli)
• Bacterial genomes = 0.6–9.4 Mbp
– Genome of bacteria usually circular
• Seldom linear, segmented
Bacterial Genetic Organization
• E. coli genome
– regulatory
• promoter/operator, signal
sequences
– coding sequences
– Average 1000 bases per bacterial
gene
– Organized on both strands
– Operons and regulons
– Monocistron vs Polycistron
organization
– Overlapping genes => ribosomal
frameshifting
Overlapping genes
Met Pro Gln
Pro Lys Trp Thr Lys Ile Cys Ser Leu His
ATGCCCCAA---//---CCAAAATGAACGAAAATCTGTTCGCTTCAT
Met Asn Glu Asn Leu Phe Ala Ser
DNA is an antiparallel double helix
• Geometry of bases and their spacial
arrangement to form H-bond cause helix
structure of dsDNA
Major groove
• B-form DNA
• pairing bases stack at the centre
• backbone intertwined
• creates minor and major grooves
34 A
• 0.34 nm (3.4 A) rise per base pair
• one full helix turn houses 10 nucleotides
20 A
DNA is an antiparallel double helix
Major groove
34 A
20 A
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DNA Is Packed to Fit the Cell
DNA Is Packed to Fit the Cell
Nucleoid of E. coli
Circle of dsDNA 1500x the
size of the cell
• Multiple loops held by
anchoring proteins
• Each loop has coiled DNA
Supercoiling Compacts DNA
• Unsupercoiled DNA = 1 winding for 10 bases
• Positive supercoils
– Winding more frequently
• Overwinding
• Negative supercoils
– Winding less frequently
• Underwinding
• Supercoils twist DNA
• Why supercoils are important?
– Eubacteria => less frequent winding
– Extreme thermophiles => more frequent winding
Relevance to Research
1
2
3
Circular
Linear
Super-coiled
Topoisomerases Regulate Supercoils
• Type I Topoisomerases
– Relieve torsional stress caused by
supercoils
– Act on one strand, How?
• Type II Topoisomerases (DNA gyrase)
– Unwind dsDNA
– Introduce negative supercoils
– Act on both strands of dsDNA, How?
• Archaeal topoisomerases
– Reverse topoisomerases
– Introduce positive supercoils
Topoisomerase I
• Single protein, nicks one strand
• Allows passages of the other strand through single strand
break
• Releaves accumulated positive supercoils ahead of
replicating DNA
Topoisomerase II (DNA Gyrase)
• two subunits, GyrB and GyrA
• GyrB binds DNA, passes to GyrA
• GyrA introduces double strand break
– 2 ATP hydrolysed
– Remains transiently attached
• Passes other dsDNA through break
• Reseals the ds break
• A negative writhe introduced
Mechanochemical analysis of DNA gyrase
Topoisomerases Regulate Supercoils
Summary Animation: Topoisomerases I and II
DNA Replication
Semiconservative replication
• Copies information from one strand to a new,
complementary strand
– Dividing cells receive one parental strand and one
newly synthesized strand
– Melt double-stranded DNA
– Polymerize new strand complementary to each melted
single strand
Replication Begins at oriC
oriC
ter
‘13-mers’
‘9-mers’
E. coli oriC: 245 bp
Replication Begins at oriC
• Timing: Dam methylation at
A of GATC (ie. GAN6mTC)
• SeqA binds to hemi
methylated duplex at OriC
• Full methylation following
cell division and loss of
SeqA affinity
• DnaA concentration rises
• Binds to 9-mer repeats at
OriC
OriC: 245 bp contining 9-mer repeats, with 13-mer
repeats in between
DnaA binding, strand melting at 13-mer by RNAP
DNA Helicase Melts DNA
• Helicase Loader (DnaC) places helicase (DnaB) at
each end of origin
Helicase
Loader
Origin
Helicase Recruits Primase
• Primase begins replication
• RNA primer forms 3OH for DNA to attach
– Evolutionary remnant?
– 1st cells thought to use RNA, not DNA
Helicase
Primase
Primosome
Primer Recruits Clamp Loader to Each Strand
• Sliding clamp binds DNA polymerase III to
each strand
DNA Pol III
Sliding
Clamp
Clamp Loader
DNA Pol III
Polymerase Proceeds 5 3 on Each Strand
•
Energy for polymerization comes from
phosphate groups on added base.
– Must add new base to 3OH of a chain
– New nucleic acids grow to extend 3 end
Each Fork Has Two Strands
• Steady growth of new “leading” strand
– Leading strand follows helicase
• Lagging strand: discontinuous, needs intermittent
release and reloading of replisome
Leading Strand
Leading
Strand
Lagging Strand
Lagging Strand Growth
• Polymerase continues to
previous primer
• Clamp loader places
primase on new site
• DNA present in 1000 base
pieces
– Okazaki fragments
RNase H Removes Primers
• One primer for each
leading strand
• Many primers on
lagging strands
– One per Okazaki
fragment
• Gaps filled in by DNA
Polymerase I
• Ligase seals nicks
DNA Replication: Sliding model
• Replisome anchored to
membrane at mid-cell
• DNA spools through as
replicated
• Proof?
• PolC-GFP stays at equator
attached to membrane
• DAPI stained DNA:
throughout cytoplasm
Animation: Summary of DNA Replication
Relevance to Research
• DNA replication in vitro
• Polymerase chain reaction (PCR)
– Amplifies specific genes from a given genome
– Need: template DNA, primers, dNTPs, DNA Polymerase,
buffer, Mg2+ fd
– Denaturation, Annealing, Elongation
PCR cycles
10
20
30 40
Animation: Proof reading function of Pol III
Both Forks Move to ter Sites
• Movement is simultaneous
• Opposite directions until both meet again at
terminus
• Replisome disassembles at ter sites
Plasmids
• Extrachromosomal pieces of DNA
• Low-copy-number plasmids
– One or two copies per cell
– Segregate similarly to chromosome
• High-copy-number plasmids
– Up to 700 copies per cell
– Divide continuously
– Randomly segregate to daughter cells
Plasmid Genes
• Advantageous under special conditions
– Antibiotic-resistance genes
– Genes encoding resistance to toxic metals
– Genes encoding proteins to metabolize rare food
sources
– Virulence genes to allow pathogenesis
– Genes to allow symbiosis
Relevance to Research
• Molecular cloning
– Plasmids are used
to import a
segment of
exogenous DNA
into a host cell.
Plasmid Replication
Bidirectional replication
Similar to chromosomal replication
Unidirectional (“rolling circle”) replication
Starts at nick bound
by RepA protein
Provides 3OH for replication
Helicase moves around
plasmid repeatedly
Complementary strand
synthesized
Used by many bacteriophages
Animation: Rolling circle replication