Bio3124Lecture 8

• 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

DNA Contains Cell InformationDNA Contains Cell Information

Bacterial Genetic OrganizationBacterial 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

ATGCCCCAA---//---CCAAAATGAACGAAAATCTGTTCGCTTCAT

Met Pro Gln Pro Lys Trp Thr Lys Ile Cys Ser Leu His

Met Asn Glu Asn Leu Phe Ala Ser

Overlapping genesOverlapping genes

DNA is an antiparallel double helixDNA 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

34 A

Major groove

20 A

DNA is an antiparallel double helix

34 A

Major groove

20 A

DNA Is Packed to Fit the CellDNA Is Packed to Fit the Cell6

DNA Is Packed to Fit the CellDNA Is Packed to Fit the Cell

• Multiple loops held by anchoring proteins

• Each loop has coiled DNA

Nucleoid of E. coli Circle of dsDNA 1500x the

size of the cell

Supercoiling Compacts DNASupercoiling 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

CircularLinear

Super-coiled

Ladder

1 2 3

Relevance to ResearchRelevance to Research

Topoisomerases Regulate SupercoilsTopoisomerases 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 ITopoisomerase 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)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 SupercoilsTopoisomerases Regulate Supercoils

Summary Animation: Topoisomerases I and II Summary Animation: Topoisomerases I and II

DNA ReplicationDNA ReplicationSemiconservative 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

oriC

ter

‘9-mers’ ‘13-mers’

Replication Begins at Replication Begins at oriCoriC

E. coli oriC: 245 bp

Replication Begins at Replication Begins at oriCoriC• 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

OriCOriC: 245 bp contining 9-mer repeats, with 13-mer repeats in betweenDnaA binding, strand melting at 13-mer by RNAP

DNA Helicase Melts DNA• Helicase Loader (DnaC) places helicase (DnaB) at

each end of origin

Helicase

Origin

Loader

Helicase Recruits PrimaseHelicase 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 StrandPrimer Recruits Clamp Loader to Each Strand

• Sliding clamp binds DNA polymerase III to each strand

DNA Pol III

DNA Pol III

SlidingClamp

Clamp Loader

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

LeadingStrand

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 modelDNA 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:Animation: Summary of DNA Replication Summary of DNA Replication

10 20 30 40

PCR cycles

• 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

Relevance to ResearchRelevance to Research

Animation:Animation: Proof reading function of Pol III Proof reading function of Pol III

Both Forks Move to Both Forks Move to terter Sites Sites

• Movement is simultaneous• Opposite directions until both meet again at

terminus• Replisome disassembles at ter sites

PlasmidsPlasmids

• 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 ResearchRelevance to Research

• Molecular cloning– Plasmids are used

to import a segment of exogenous DNA into a host cell.

Plasmid ReplicationPlasmid Replication Bidirectional replication

Similar to chromosomal replication Unidirectional (“rolling circle”) replication

Starts at nick bound by RepA proteinProvides 3OH for replicationHelicase moves around

plasmid repeatedlyComplementary strand

synthesizedUsed by many bacteriophages

Animation: Rolling circle replication Animation: Rolling circle replication