slide1 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
DNA polymerase summary PowerPoint Presentation
Download Presentation
DNA polymerase summary

Loading in 2 Seconds...

play fullscreen
1 / 43

DNA polymerase summary - PowerPoint PPT Presentation


  • 127 Views
  • Uploaded on

DNA polymerase summary. DNA replication is semi-conservative. DNA polymerase enzymes are specialized for different functions. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease. DNA polymerase structures are conserved.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'DNA polymerase summary' - errin


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
dna polymerase summary
DNA polymerase summary

DNA replication is semi-conservative.

DNA polymerase enzymes are specialized for different functions.

DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease.

DNA polymerase structures are conserved.

But: Pol can’t start and only synthesizes DNA 5’-->3’!

Editing (proofreading) by 3’-->5’ exo reduces errors.

High fidelity is due to the race between addition and editing.

Mismatches disfavor addition by DNA pol I at 5 successive positions. The error rate is ~1/109.

replication fork summary
Replication fork summary
  • DNA polymerase can’t replicate a genome.
          • ProblemSolution ATP?
  • No single stranded template Helicase +
  • The ss template is unstable SSB (RPA (euks)) -
  • No primer Primase (+)
  • No 3’-->5’ polymerase Replication fork
  • Too slow and distributive SSB and sliding clamp-

2. Replication fork is organized around an asymmetric, DNA- polymerase III dimer.

3. Both strands made 5’-->3’.

4. “Leading strand” is continuous; “lagging strand” is discontinuous.

dna polymerase can t replicate a genome
DNA polymerase can’t replicate a genome!

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase

Too slow and distributive

solution the replication fork
Solution: the replication fork

No single-stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase

Too slow and distributive

Schematic drawing of a replication fork

dna replication factors were discovered using temperature sensitive mutations
DNA replication factors were discovered using “temperature sensitive” mutations

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

37 ºC

Mutations that inactivate the DNA replication machinery are lethal.

Temperature sensitive (conditional) mutations allow isolation of mutations in essential genes.

42 ºC

42 ºC,

Mutant gene

overexpressed

a hexameric replicative helicase unwinds dna ahead of the replication fork
A hexameric replicative helicase unwinds DNA ahead of the replication fork

Helicase assay

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

ds DNA

Replicative DNA helicase is called DnaB in E. coli.

DnaB couples ATP binding and hydrolysis to DNA strand separation.

ss DNA

ssb or rpa cooperatively binds ss dna template
SSB (or RPA) cooperatively binds ss DNA template

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

SSB (single-strand binding protein (bacteria)) or RPA (Replication Protein A (eukaryotes)):

No ATP used.

Filament is substrate for DNA pol.

ss DNA + SSB

ds DNA

ssb tetramer structure
SSB tetramer structure

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

SSB (bacteria) and RPA (eukaryotes)

form tetramers.

The C-terminus of SSB binds replication factors (primase, clamp loader (chi subunit))

C

C

N

N

N

N

ss DNA + SSB

C

C

Conservation

Positive potential

ds DNA

slide11

DNA synthesis is primed by a short RNA segment

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

Primase: DNA-dependent RNA polymerase

Primase makes about 10-base RNA.

The product is a RNA/DNA hybrid.

RNA primer has a free 3’OH.

Start preference for CTG on template

Uses ATP, which ends up across from T in the RNA/DNA hybrid.

dnag primase defines a distinct polymerase family dna dependent rna pol
DnaG primase defines a distinct polymerase family (DNA dependent RNA pol)

Model of “primosome”:

DnaB helicase +DnaG primase

Ribbon

diagram

DnaB helicase

Map of

surface charge

DnaG primase

primase passes the primed template to dna polymerase
Primase passes the primed template to DNA polymerase

Leading strand: continuous

Lagging strand: discontinuous

slide14

DNA pol III “holoenzyme” is asymmetric

DNA pol III holoenzyme:

A molecular machine

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

Synthesizes

Leading

Strand

Synthesizes

Lagging

Strand

binds SSB

 opens clamp ()

replication fork
Replication fork

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase

Too slow and distributive

replication fork17
Replication fork

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase

Too slow and distributive

synthesis of okazaki fragments by pol iii holoenzyme
Synthesis of Okazaki fragments by pol III holoenzyme

When pol III reaches the primer of the previous Okazaki fragment, clamp loader removes 2 from the DNA template. As a result, the pol III on the lagging strand falls off the template.

Clamp loader places 2 on the next primer-template.

replication fork summary22
Replication fork summary
  • DNA polymerase can’t replicate a genome.
          • Solution ATP?
  • No single stranded template Helicase +
  • The ss template is unstable SSB (RPA (euks)) -
  • No primer Primase (+)
  • No 3’-->5’ polymerase Replication fork
  • Too slow and distributive SSB and sliding clamp-

2. Replication fork is organized around an asymmetric, DNA- polymerase III dimer.

3. Both strands made 5’-->3’.

4. “Leading strand” is continuous; “lagging strand” is discontinuous.

replication fork summary23
Replication fork summary
  • DNA polymerase can’t replicate a genome.
          • ProblemSolution ATP?
  • No single stranded template Helicase +
  • The ss template is unstable SSB (RPA (euks)) -
  • No primer Primase (+)
  • No 3’-->5’ polymerase Replication fork
  • Too slow and distributive SSB and sliding clamp -
  • Sliding clamp can’t get on Clamp loader (/RFC) +

Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -

Lagging strand is nicked DNA ligase +

Helicase introduces positive Topoisomerase II +

supercoils

2. DNA replication is fast and processive

rfc clamp loader complex puts the clamp on dna
/RFC clamp loader complex puts the clamp on DNA

 complex -- bacteria

RFC -- eukaryotes

Sliding clamp can’t get on

Lagging strand contains RNA

Lagging strand is nicked

Helicase introduces + supercoils

(Replication Factor C)

rfc reaction
RFC reaction

RFC + clamp + ATP opens clamp

Ternary complex + DNA/RNA --> Closed clamp + RFC + ADP + Pi

schematic drawing of the rfc pcna complex on the primer template
Schematic drawing of the RFC:PCNA complex on the primer:template

RFC contains 5 similar subunits that spiral around DNA.

The RFC helix tracks the DNA or DNA/RNA helix

RFC

PCNA

DNA:RNA

rfc pcna crystal structure
RFC:PCNA crystal structure

RFC

PCNA

DNA:RNA

RFC:PCNA crystal structure

ssb opens hairpins maintains processivity and mediates exchange of factors on the lagging strand
SSB opens hairpins, maintains processivity andmediates exchange of factors on the lagging strand

No single stranded template

The ss template is unstable

No primer

No 3’-->5’ polymerase.

Too slow in vitro.

SSB (bacteria) and RPA (eukaryotes)

form tetramers.

The C-terminus of SSB binds replication factors (Primase, Clamp loader (chi subunit))

SSB:DNA

binds primase

Primer:template:SSB

Binds clamp loader

Clamp loader exchanges

with pol III on the clamp

Primase - to - pol III switch

dna polymerase 5 3 exonuclease or rnase h remove rna primers
DNA polymerase 5’-->3’ exonuclease or RNase Hremove RNA primers

Sliding clamp can’t get on

Lagging strand contains RNA

Lagging strand is nicked

Helicase introduces + supercoils

DNA polymerase I 5’-->3’ exo creates ss template.

Pol works on the PREVIOUS Okazaki fragment!

OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs

primer

dna polymerase 5 3 exonuclease or rnase h remove rna primers32
DNA polymerase 5’-->3’ exonuclease or RNase Hremove RNA primers

Sliding clamp can’t get on

Lagging strand contains RNA

Lagging strand is nicked

Helicase introduces + supercoils

DNA polymerase I 5’-->3’ exo creates ss template.

Pol works on the PREVIOUS Okazaki fragment!

OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs

primer

dna ligase seals the nicks
DNA ligase seals the nicks

Adenylylate the

enzyme

2. Transfer AMP to

the PO4 at the nick

3. Seal nick, releasing

AMP

Three steps in the DNA ligase reaction

all tied up in knots
All tied up in knots

Sliding clamp can’t get on

Lagging strand contains RNA

Lagging strand is nicked

Helicase introduces + supercoils

topological problems in dna can be lethal

Gene misexpression

  • Chromosome breakage
  • Cell death
“Topological” problems in DNA can be lethal

(+) supercoils

(-) supercoils

(+) supercoils

precatenanes

catenanes

slide37

Relaxed/disentangled

Topoisomerases control chromosome topology

Catenanes/knots

Topos

  • Major therapeutic target - chemotherapeutics/antibacterials
  • Type II topos transport one DNA through another
topoisomerases cut one strand i or two ii
Topoisomerases cut one strand (I) or two (II)

Topoisomerase I - Cuts ssDNA region (1A (proks)) or nicks DNA (1B (euks))

Topoisomerase II - Cuts DNA and passes one duplex through the other!

topoisomerase ii is a dimer that makes two staggered cuts
Topoisomerase II is a dimer that makes two staggered cuts

Tyr OH attacks PO4 and forms a covalent intermediate

Structural changes in the protein open the gap by 20 Å!

slide40

Type IIA topoisomerases comprise a homologous superfamily

ATPase

DNA Binding/Cleavage

GyrB

GyrA

Gyrase

(proks)

Topo II

(euks)

type iia topoisomerase mechanism

ADP

Type IIA topoisomerase mechanism

T-segment

G-segment

1

2

4

3

  • “Two-gate” mechanism
  • Why is the reaction directional?
  • What are the distinct conformational states?
summary of the replication fork42
Summary of the replication fork

“Fingers”

“Thumb”

“Palm”

accessory factors summary
Accessory factors summary
  • DNA polymerase can’t replicate a genome.
          • Solution ATP?
  • No single stranded template Helicase +
  • The ss template is unstable SSB (RPA (euks)) -
  • No primer Primase (+)
  • No 3’-->5’ polymerase Replication fork
  • Too slow and distributive SSB and sliding clamp -
  • Sliding clamp can’t get on Clamp loader (/RFC) +

Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -

Lagging strand is nicked DNA ligase +

Helicase introduces positive Topoisomerase II +

supercoils

2. DNA replication is fast and processive