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Bio3124 Lecture 8. Bacterial Genetics: I. Genome replication and packing. 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

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bio3124 lecture 8

Bio3124Lecture 8

Bacterial Genetics:I. Genome replication and packing

dna contains cell information
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 o rganization
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

Bacterial Genetic Organization
overlapping genes
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
DNA is an antiparallel double helix
  • Geometry of bases and their spacial arrangement to form H-bond cause helix structure of dsDNA
  • B-form DNA
  • pairing bases stack at the centre
  • backbone intertwined
  • creates minor and major grooves
  • 0.34 nm (3.4 A) rise per base pair
  • one full helix turn houses 10 nucleotides

Major groove

34 A

20 A

dna is packed to fit the cell1
Multiple loops held by anchoring proteins

Each loop has coiled DNA

DNA Is Packed to Fit the Cell
  • Nucleoid of E. coli
  • Circle of dsDNA 1500x the size of the cell
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

Supercoiling Compacts DNA
relevance to research
Relevance to Research

Ladder

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?

Archaealtopoisomerases

Reverse topoisomerases

Introduce positive supercoils

Topoisomerases Regulate Supercoils
topoisomerase i
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

Topoisomerase II (DNA Gyrase)

Mechanochemical analysis of DNA gyrase

dna replication
Semiconservativereplication

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

DNA Replication
replication begins at oric
Replication Begins at oriC

oriC

ter

‘13-mers’

‘9-mers’

E. coli oriC: 245 bp

replication begins at oric1
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

Replication Begins at oriC

OriC: 245 bp contining 9-mer repeats, with 13-mer repeats in between

DnaA binding, strand melting at 13-mer by RNAP

helicase recruits primase
Primase begins replication

RNA primer forms 3OH for DNA to attach

Evolutionary remnant?

1st cells thought to use RNA, not DNA

Helicase Recruits Primase

Helicase

Primase

Primosome

polymerase proceeds 5 3 on each strand
Energy for polymerization comes from phosphate groups on added base.

Must add new base to 3OH of a chain

New nucleic acids grow to extend 3 end

Polymerase Proceeds 5 3 on Each Strand
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

Each Fork Has Two Strands

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

Lagging Strand Growth
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

RNase H Removes Primers
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

DNA Replication: Sliding model
relevance to research1
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

both forks move to ter sites
Movement is simultaneous

Opposite directions until both meet again at terminus

Replisome disassembles at ter sites

Both Forks Move to 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

Plasmids
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

Plasmid Genes
relevance to research2
Relevance to Research
  • Molecular cloning
    • Plasmids are used to import a segment of exogenous DNA into a host cell.
plasmid replication
Plasmid Replication
  • Bidirectional replication
    • Similar to chromosomal replication
  • Unidirectional (“rolling circle”) replication
    • Starts at nick bound by RepA protein
    • Provides 3OH for replication
    • Helicase moves around plasmid repeatedly
    • Complementary strand synthesized
    • Used by many bacteriophages
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