TR056/PG1005
Lecture 15
DNA Replication and Mitosis
Dr. Neil Docherty
My Teaching Objectives
• Explain how the DNA code is accessed for DNA
replication
• Provide an overview of the sequential events of
DNA replication
• Highlight the importance of final editing of
replication
p
fidelityy and the requirements
q
for transition
to the mitotic phase of the cell life cycle
DNA Replication-Problem 1 Access
Nuclesome formation compact DNA seven fold
H li l stacking
Helical
t ki off solenoids
l
id provides
id up tto 104 fold
f ld condensation.
d
ti
Now DNA resembles
packets=chromosomes.
Mostt prominent
M
i
t in
i
duplicated form during
mitotic metaphase
metaphase.
Accessing Supercoiled DNA
Topoisomerase enzymes
Nuclease/ligase activity
Single stranded
Relaxation of wound
structure
Double stranded
Passing of double strands
out and through
g coils
ACCESSIBILITY OF CHROMATIN
TO REPLICATION MACHINERY
Opening The Helix
• Started by the activity of initiator proteins-helicase
• Bind
Bi d DNA and
db
break
kh
hydrogen
d
b
bonds
d att replication
li ti origins
i i
•
These sites are rich in A-T repeats, as A-T has 2 rather than 3
hydrogen bonds
• Numerous such sites exist along the length of the chromosome
• Replication proceeds in both directions and copies each strand
• Above 2 points reduce total copying time!
3’ Initiator protein action
5’
5’
3’
5’
3’
T A rich replication origin
T-A
Single strand
3’
3
5’
•Unzipped strands held in single strand conformation
by single strand binding protein
Bidirectional Extension
DNA Polymerase
•
It is really a large complex of distinct
DNA synthesis enzymes.
•
Major feature is the synthesis of DNA
in a 5’ to 3’ direction
•
Function is to add
deoxyribonucleotide to 3’ end of
nascent strand by the formation of a
phosphodiester bond
•
Requires a primer strand with free 3’OH group
KEY
REACTION
RNA Primer Synthesis
• Catalysed by primase enzyme (RNA polymerase)
• Uses single stranded DNA as a template for the
formation of a short stretch (10 nt.) long of double
strand
t dh
hybrid
b id
• Provides a free 3’-OH for polymerisation to proceed
Directionality (5’-3’) of DNA Polymerisation
Leads To Designation of Leading and
Lagging Strands
Synthesis from leading strand occurs continuously from RNA primer
Synthesis from lagging strand is discontinous via backstitching of
Okazaki fragments.
DNA Polymerase 3’-5’
3 -5 Proofreading
• Only one error occurs per 107 nucleotide pairs copied
• Due to 3’ to 5’ exonuclease activity of DNA polymerase
• Removal of mismatched base-pairs
Removal of Primers
• 5’-3’ ribonuclease activity of DNA polymerase
• Lagging strand gaps then filled by DNA repair
polymerase
• Double
Do ble heli
helix sealed b
by DNA ligase
Post-Replication
• Compaction of DNA into heterochromatin for mitotic
cell division
Mitosis and Cytokinesis Overview
-M stage
g of cell
cycle
-7 substages
The Microtubules and
Chromosomes Visualised
Your Learning Objectives
Your learning from today should focus on being able to;
• Explain how and why the DNA molecules require to
be decondensed for DNA replication to proceed
• Discuss DNA synthesis and editing from the leading
and lagging strands
• Provide an overview of the cytoskeletal changes that
underpin
p the p
partition of the replicated
p
chromosomes
during the mitotic phase of the cell life cycle