Biochimica e biologia molecolare - duplicazione del DNA

Duplication e struttura del DNA

Organizzazione del cromosoma

Gli istoniglobular corehistone H111 nmnucleosomeH1 si legano a specifiche regioni del nucleosoma. Il packaging del nucleosoma è mediato dall'istone H1.

• Short region of DNA double helix: 2 nm
• "Beads-on-a-string" form of chromatin: 11 nm
• 30-nm chromatin fiber of packed nucleosomes: 30 nm
• Section of chromosome in an extended form: 300 nm
• Condensed section of metaphase chromosome: 700 nm
• Entire metaphase chromosome: 1400 nm

Questo cromosoma si vede solo nelle cellule in duplicazione.

Meccanismo di duplicazione del DNA

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.

Origine e crescita della replicazione

First replication (³H), Second replication No ³H

L’origine di replicazione

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

La forca di replicazione è bidirezionale.

Autoradiogramma e interpretazione

• (a) Predicted fiber autoradiographic pattern
• Hot Warm
• Unidirectional growth OR
• Bidirectional growth

Un meccanismo solo è quello corretto.

La DNA Polimerasi

(A) Incoming deoxyribonucleoside triphosphate

• Primer strand
• Template strand
• 5'-to-3' direction of chain growth

(B) Incoming deoxyribonucleoside triphosphate

• Repaired DNA helix
• Template strand
• Gap in helix

La correzione delle bozze

Attività polimerasica: 5’→3’
Attività esonucleasica: 3’→5’

I filamenti guida e tardivo

La DNA ligasi ripara i legami fosfodiesterici rotti.

Step 1
Step 2

L'inizio della duplicazione

• 9-mers
• 13-mers
• Negatively supercoiled template
• DnaA
• ATP
• Initial complex
• Open complex
• ATP
• DnaC
• DnaB (helicase)
• ATP
• Prepriming complex
• DnaB

La DNA elicasi e le proteine che legano il filamento singolo.

La primasi e 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, β-subunit clamp, Core of DNA polymerase III

Newly formed DNA strand, Template DNA strand

La pinza β

• DNA polymerase
• Two halves of sliding clamp
• Clamped polymerase tethered on DNA
• Parental duplex
• Fork
• Core 1b clamp
• Direction of growth of leading strand
• Direction of fork movement
• Leading strand, Lagging strand
• Direction of DnaB movement
• DnaB helicase
• Primase
• Primer
• Core 2
• Direction of growth of lagging strand
• SSB protein

La duplicazione crea "tensione"

• Rapid rotation of the DNA helix needed here
• Leading-strand template
• Lagging-strand template
• DNA polymerase on leading strand
• Newly synthesized DNA chain
• One end of the DNA double helix cannot rotate relative to the other end
• Type I 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
• RNA primer, Lagging-strand template
• 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?