1

TheMolecularBasisofInheritanceBIOL100

Ch.11

Overview:Life’sOpera9ngInstruc9ons

•  JamesWatsonandFrancisCrick

q  1953-produceddouble-helicalmodelforthestructureofDNA

StructuralModelofDNA•  RosalindFranklinandMauriceWilkins

q  X-raycrystallography

•  FranklinproducedapictureofDNA

(a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

2

•  WatsonandCrick

q  basespairedlikewithlike

•  resultedinanon-uniformwidth

q  purinewithapyrimidine

•  uniformwidthconsistentwithX-ray

StructuralModelofDNA

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

•  ThereforeWatsonandCrickrealized…

q  pairingmorespecific

•  dictatedbythebasestructures

q  Determinedadenine(A)withthymine(T)

•  guanine(G)withcytosine(C)

o  explainsChargaff’srule:

Ø  inanyorganismtheamountofA=T

•  andtheamountofG=C

StructuralModelofDNA

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

Fig.16-7

(c) Space-filling model

Hydrogen bond 3ʹ end

5ʹ end

3.4 nm

0.34 nm 3ʹ end

5ʹ end

(b) Partial chemical structure (a) Key features of DNA structure

1 nm

3

Fig.16-9-1

A T

G C

T A

T A

G C

(a) Parent molecule

Fig.16-9-2

A T

G C

T A

T A

G C

A T

G C

T A

T A G C

(a) Parent molecule (b) Separation of strands

Fig.16-9-3

A T

G C

T A

T A

G C

(a) Parent molecule

A T

G C

T A

T A G C

(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand

(b) Separation of strands

A T

G C

T A

T A

G C

A T

G C

T A

T A G C

4

•  Semiconserva9vemodel

q  Eachdaughtermolecule

•  oneold,onenewstrand

•  CompeYngmodels

q  conservaYvemodel

•  thetwoparentstrandsrejoin

q  dispersivemodel

•  eachstrandisamixofoldandnew

DNAReplica9on

Parent cell First replication

Second replication

(a) Conservative model

(b) Semiconserva- tive model

(c) Dispersive model

DNAReplica9on•  DNAreplicaYon

q  remarkablespeedandaccuracy

•  MorethanadozenenzymesandotherproteinsparYcipate

DNAReplica9on

Origin of replication Parental (template) strand

Daughter (new) strand

Replication fork

Replication bubble

Two daughter DNA molecules

(a) Origins of replication in E. coli

Origin of replication Double-stranded DNA molecule

Parental (template) strand Daughter (new) strand

Bubble Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

0.5 µm

0.25 µm

Double- stranded DNA molecule

•  Originsofreplica9on

q  SiteswheretwoDNAstrandsseparate

•  opensreplicaYon“bubble”

q  eukaryoYcchromosome

•  hundreds-thousandsoforiginsofreplicaYon

q  ReplicaYonproceedsinbothdirecYonsfromeach

origin

•  unYltheenYremoleculeiscopied

DNAReplica9on

5

Topoisomerase

Helicase

Primase Single-strand binding proteins

RNA primer

5ʹ 5ʹ

5ʹ 3ʹ

3ʹ

3ʹ

•  Replica9onfork

q  Y-shapedregion

•  endofeachreplicaYon

bubble

o  wherenewDNAstrands

areelongaYng

•  Helicases

q  untwistDNAatreplicaYonforks

DNAReplica9on

Overview Origin of replication

Leading strand

Leading strand

Lagging strand

Lagging strand Overall directions

of replication

Leading strand

Lagging strand

Helicase

Parental DNA

DNA pol III

Primer Primase

DNA ligase

DNA pol III

DNA pol I

Single-strand binding protein

5ʹ

3ʹ

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3 ́1 3 2

4

•  Primer

q  ShortRNAstrandrequired

foriniYaYon

•  Primase

q  EnzymethatstartsanRNA

chainfromscratch

•  addsRNAnucleoYdes

o  UsesDNAasa

template

•  5–10nucleoYdeslong

o  3ʹendstarYng

pointfornewDNA

DNAReplica9on

Fig.16-14

A

C

T

G

G

G

G C

C C

C

C

A

A

A T

T

T

New strand 5ʹ end

Template strand 3ʹ end 5ʹ end 3ʹ end

3ʹ end

5ʹ end 5ʹ end

3ʹ end

Base Sugar

Phosphate

Nucleoside triphosphate

Pyrophosphate

DNA polymerase

6

Fig.16-15

Leading strand

Overview Origin of replication

Lagging strand

Leading strand Lagging strand

Primer

Overall directions of replication

Origin of replication

RNA primer “Sliding clamp”

DNA poll III Parental DNA

5 ́

3 ́

3 ́

3 ́

3 ́

5 ́

5 ́

5 ́

5 ́

5 ́

•  laggingstrand

q  Strandconstructedawayfrom

replicaYonfork

•  Okazakifragments

q  seriesofsegments

q  joinedtogetherbyDNAligase

An9parallelElonga9on

Fig.16-16a

Overview Origin of replication

Leading strand

Leading strand

Lagging strand

Lagging strand

Overall directions of replication

1 2

7

Fig.16-17

Overview Origin of replication

Leading strand

Leading strand

Lagging strand

Lagging strand Overall directions

of replication

Leading strand

Lagging strand

Helicase

Parental DNA

DNA pol III

Primer Primase

DNA ligase

DNA pol III

DNA pol I

Single-strand binding protein

5ʹ 3ʹ

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ 1 3 2

4

TheDNAReplica9onComplex•  DNAreplicaYoncomplex

•  proteinsthatparYcipateinreplicaYon

•  “DNAreplicaYonmachine”

•  probablystaYonaryduringthereplicaYonprocess

•  Recentstudies

•  supportamodelinwhichDNApolymerasemolecules“reelin”parentalDNA

•  and“extrude”newlymadedaughterDNAmolecules

DNA Replication

ProofreadingandRepairingDNA•  Proofreading

q  EukaryoYcDNApolymerases

•  replaceanyincorrectnucleoYdes

•  Mismatchrepair

q  repairenzymescorrecterrorsinbasepairing

•  DNAcanbedamaged

q  bychemicals,radioacYveemissions,X-rays,UVlight,andothermolecules(incigarecesmokeforexample)

•  Nucleo9deexcisionrepair

q  Nuclease,polymerase,ligase

•  cutout(nuclease),replace(polymerase)damagedstretchesofDNA,andconnect(ligase)newnucleoYdestopre-exisYngstrand

8

Fig.16-18

Nuclease

DNA polymerase

DNA ligase

Thyminedimer

Replica9ngtheEndsofDNAMolecules

•  LimitaYonsofDNApolymerase

q  createproblemsforthelinearDNAofeukaryoYcchromosomes

q  StandardreplicaYonmachinery

•  providesnowaytocompletethe5ʹends

•  repeatedroundsofreplicaYon

o  produceshorterDNAmolecules

Ends of parental DNA strands

Leading strand Lagging strand

Lagging strand

Last fragment Previous fragment

Parental strand

RNA primer

Removal of primers and replacement with DNA where a 3ʹ end is available

Second round of replication

New leading strand

New lagging strand

Further rounds of replication

Shorter and shorter daughter molecules

5 ́

3 ́

3 ́

3 ́

3 ́

3 ́

5 ́

5 ́

5 ́

5 ́

•  Telomeres

q  NucleoYdesequences

•  AtendsofeukaryoYcchromosomalDNA

molecules

q  DonotpreventtheshorteningofDNAmolecules

•  PostponetheerosionofgenesneartheendsofDNAmolecules

•  Ithasbeenproposedthattheshorteningoftelomeresisconnectedtoaging

Replica9ngtheEndsofDNAMolecules

1 µm

9

•  Thenwhataboutgametes?

q  Ifgermcellchromosomesbecameshorterineverycellcycle

o  geneseventuallydamaged

Ø  andgametestheyproduce

•  telomerase

q  catalyzesthelengtheningoftelomeresingermcells

•  fountainofyouth?

o  hcp://www.nature.com/news/2010/101128/full/news.2010.635.html

Replica9ngtheEndsofDNAMolecules

•  Theshorteningoftelomeres

q  mightprotectcellsfromcancerousgrowth

•  bylimiYngthenumberofcelldivisions

•  evidenceoftelomeraseacYvityincancercells

q  mayallowcancercellstopersist

Replica9ngtheEndsofDNAMolecules

DNApackaging

•  Bacterialchromosome

q  Circular,smallamountofprotein

q  supercoiled,innucleoidregion

•  EukaryoYcchromosome

q  Linear,largeamountofprotein

PLAY

10

•  Chroma9n

q  complexofDNAandprotein

q  Histones

•  proteinsthatareresponsibleforthefirstlevelofDNApackinginchromaYn

DNApackaging

Fig.16-21a

DNA double helix (2 nm in diameter)

Nucleosome (10 nm in diameter)

Histones Histone tail H1

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

•  ChromaYnisorganizedintofibers

•  10-nmfiber

•  DNAwindsaroundhistonestoformnucleosome“beads”

•  NucleosomesarestrungtogetherlikebeadsonastringbylinkerDNA

•  30-nmfiber

•  InteracYonsbetweennucleosomescausethethinfibertocoilorfoldintothisthickerfiber

DNApackaging

•  300-nmfiber

q  The30-nmfiberforms

loopeddomainsthat

acachtoproteins

•  Metaphasechromosome

q  Loopeddomains

coilfurther

q  WidthofachromaYd

is700nm

DNApackaging

30-nm fiber

Chromatid (700 nm)

Loops Scaffold

300-nm fiber

Replicated chromosome (1,400 nm)

30-nm fiber

Looped domains (300-nm fiber)

Metaphase chromosome

11

•  MostchromaYn

q  looselypackedduringinterphase

•  condensespriortomitosis

•  Euchroma9n

q  LooselypackedchromaYn

•  Heterochroma9n

q  HighlycondensedchromaYn

•  centromeresandtelomeres

q  Duringinterphase

•  DensepackingoftheheterochromaYnmakesitdifficultforthecelltoexpressgeneYcinformaYoncodedintheseregions

DNApackaging

Youshouldnowbeableto:

1.  DescribethestructureofDNA

2.  DescribetheprocessofDNAreplicaYon;includethefollowingterms:anYparallelstructure,DNApolymerase,leadingstrand,laggingstrand,Okazakifragments,DNAligase,primer,primase,helicase,topoisomerase

3.  DescribethefuncYonoftelomeres

4.  CompareabacterialchromosomeandaeukaryoYcchromosome