Bio3124 lecture 8
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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

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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 an antiparallel double helix1

DNA is an antiparallel double helix

Major groove

34 A

20 A


Dna is packed to fit the cell

6

DNA Is Packed to Fit the Cell


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


Topoisomerases regulate supercoils1

Topoisomerases Regulate Supercoils


Summary animation topoisomerases i and ii

Summary Animation: Topoisomerases I and II


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


Dna helicase melts dna

Helicase Loader (DnaC) places helicase (DnaB) at each end of origin

DNA Helicase Melts DNA

Helicase

Loader

Origin


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


Primer recruits clamp loader to each strand

Sliding clamp binds DNA polymerase III to each strand

Primer Recruits Clamp Loader 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 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


Animation summary of dna replication

Animation: Summary of DNA Replication


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


Animation proof reading function of pol iii

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

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


Animation rolling circle replication

Animation: Rolling circle replication


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