Biologia e genetica - duplicazione del DNA

Gli istoni

globular core

histone H1

nucleosome

H1 BINDS TO SPECIFIC REGION OF NUCLEOSOME

NUCLEOSOME PACKING IS MEDIATED BY HISTONE H1

Gli istoni

globular core

histone H1

nucleosome

H1 BINDS TO SPECIFIC REGION OF NUCLEOSOME

NUCLEOSOME PACKING IS MEDIATED BY HISTONE H1

short region of DNA double helix

"beads-on-a-string" form of chromatin

30-nm chromatin fiber of packed nucleosomes

section of chromosome in an extended form

condensed section of metaphase chromosome

entire metaphase chromosome

Questo cromosoma si vede solo nelle cellule in duplicazione

Come si duplica il DNA?

Conservative mechanism

Semiconservative mechanism

Old strand

New strand

Parental

First generation

Second generation

¹⁴N/¹⁴N (light) DNA

¹⁴N/¹⁵N (hybrid) DNA

¹⁵N/¹⁵N (heavy) DNA

La Duplicazione è Semiconservativa

First replication (3^H)

Second replication No 3^H

L’Origine di Replicazione

• (a) Unidirectional growth of single strands from two origins
  • Origin 1
  • Old strand
  • New strand
  • Growing point
  • Origin 2
  • 2 origins
  • 2 growing ends
• (b) Unidirectional growth of both strands from one origin
  • Growth
  • Origin
  • Growing fork
  • 1 origin
  • 1 fork
• (c) Bidirectional growth of both strands from one origin
  • Growth
  • Origin
  • Growing fork
  • 1 origin
  • 2 forks

La forca di replicazione è bidirezionale

Autoradiogram

Interpretation

(a) Predicted fiber autoradiographic pattern

Hot Warm

Unidirectional growth

ORIORI

Bidirectional growth

Un Meccanismo solo è quello corretto

La DNA Polimerasi

5'-to-3' direction of chain growth

incoming deoxyribonucleoside triphosphate

primer strand

template strand

repaired DNA helix

gap in helix

La correzione delle bozze

Attività polimerasica: 5’→3’

Attività esonucleasica: 3’→5’

I Filamenti Guida e Tardivo

lagging-strand template

leading-strand template

most recently synthesized DNA strands

La DNA ligasi

broken phosphodiester bond

STEP 1

STEP 2

L’inizio della duplicazione

9-mers

13-mers

Negatively supercoiled template

DnaA

ATP

Initial complex

Open complex

ATP

Prepriming complex

DnaC

DnaB (helicase)

DnaB

La DNA Elicasi

DNA helicase binds

Le proteine che legano il filamento singolo

DNA polymerase

5' 3'

single-stranded region of DNA template with short regions of base-paired "hairpins"

single-strand binding protein monomers

5' 3'

cooperative protein binding straightens region of chain

La Primasi

I Frammenti di Okazaki

new RNA primer synthesis by DNA primase

DNA polymerase adds to new RNA primer to start new Okazaki fragment

DNA polymerase finishes DNA fragment

old RNA primer erased and replaced by DNA

nick sealing by DNA ligase joins new Okazaki fragment to the growing chain

Lagging strand

Leading strand

DNA ligase

Okazaki fragment

La struttura fine della DNA Polimerasi

Nucleotide being added to 3' end

Direction of synthesis

3'

5'

β-subunit clamp

Core of DNA polymerase III

Newly formed DNA strand

5'

3'

Template DNA strand

La Pinza β

DNA polymerase

two halves of sliding clamp

clamped polymerase tethered on DNA

Parental duplex

5' 3'

Fork

Direction of growth of leading strand

Direction of fork movement

3' 5'

Leading strand

3' 5'

Lagging strand

Core 1

β clamp

τ

Primer

Core 2

Direction of DnaB movement

DnaB helicase

Primase

β clamp

SSB protein

Direction of growth of lagging strand

La duplicazione Crea “Tensione”

DNA polymerase on leading strand newly synthesized DNA chain

• leading-strand template
• lagging-strand template

rapid rotation of the DNA helix needed here

La Topoisomerasi

• one end of the DNA double helix cannot rotate relative to the other end
• type 1 DNA topoisomerase with tyrosine at the active site
• DNA topoisomerase covalently attaches to a DNA phosphate, thereby breaking a phosphodiester linkage in one DNA strand
• the two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain
• the original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible
• spontaneous re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase in an unchanged form

La Correzione delle Bozze

error in newly made strand

BINDING OF MISMATCH PROOFREADING PROTEINS

MutS MutL

DNA SCANNING DETECTS NICK IN NEW DNA STRAND

STRAND REMOVAL

REPAIR DNA SYNTHESIS

new RNA primer synthesis by DNA primase

DNA polymerase adds to new RNA primer to start new Okazaki fragment

DNA polymerase finishes DNA fragment

old RNA primer erased and replaced by DNA

nick sealing by DNA ligase joins new Okazaki fragment to the growing chain

Come si completa?