D N A
R E P L I C A T I O N
E l i s a H e r a w a t i , P h . D
DNA REPLICATION
• The copying of DNA is remarkable in its speed and accuracy
• More than a dozen enzymes and other proteins participate in DNA replication
• DNA is replicated during the S (synthesis) stage of the
cell cycle.
• Each body cell gets a complete set of
identical DNA.
Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”
A eukaryotic chromosome may have hundreds or even thousands of
origins of replication
DNA
Replication
Replication proceeds in both
directions from each origin, until the entire molecule is copied
Replication of DNA
• base pairing allows each strand to serve as a template for a new strand
• new strand is 1/2 parent template &
1/2 new DNA
DNA Replication
• Large team of enzymes coordinates replication
Components of Replication
▪ DNA polymerase – Deoxynucleotide polymerization
▪ Helicase – Processive unwinding of DNA
▪ Topoisomerases – Relieve torsional strain that results from helicase-induced unwinding
▪ RNA primase – Initiates synthesis of RNA primers
▪ Single-strand binding proteins – Prevent premature reannealing of dsDNA
▪ DNA ligase – Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand
DNA REPLICATION
• Parental strans are not degraded
• Base pairing allows each strand to serve as a template for a new strand
• New duplex is ½ parent template & ½ new DNA
Semi
conser vative
Semi
conser vative
DNA REPLICATION
The parent molecule directs synthesis of an entirely new double-stranded molecule, such that after one round of replication, one molecule is conserved as two old strands. This is repeated in the second round
Conser vative
DNA REPLICATION
Material in the two parental strands is distributed more or less randomly between two daughter molecules. Its distributed symmetrically between the two daughters molecules. Other distributions are possible
Dispersive
DNA REPLICATION
Fig. 16-10
Parent cell First
replication
Second replication
(a) Conservative model
(b) Semiconserva- tive model
(c) Dispersive model
DNA REPLICATION
Semi discontinuous
• Leading & Lagging strands 1. Leading strand
- Continuous synthesis 2. Lagging strand
- Okazaki fragments
- Joined by ligases
DNA Replication
Primer is needed
• DNA polymerase can only add nucleotide to 3’end of a growing DNA strand
- Need a “starter” nucleotide to make a bond
• Strand only grows 5’-3’
• Template is read in the 3’-5’ direction
while polymerization takes place in the 5’-
3’ direction
RNA Primer
PRIMER
• Synthesized by primase
• Serves as a starter sequence for DNA polymerase III
• Only one RNA Primer-required for the leading strand
• RNA Primers for the lagging strand depends on the number of “Okazaki Fragment”
• RNA Primer has a free 3’OH group to which the first nucleotide is bound
Okazaki fragment is a synthesized DNA fragment that are formed on the lagging template strand during DNA replication
They are separated by 10-nucleotide RNA primers and are unligated until RNA primers are removed, followed by enzyme ligase connecting the two Okazaki fragments into one
continuous newly synthesized complementary strand
Okazaki
Fragment
On the leading strand DNA
replication proceeds continuously along the DNA molecule, but on the lagging strand the new DNA is made in fragments, which are later joined together by a DNA ligase enzyme
This is because the enzymes that synthesize the new DNA can only work in one direction along the parent DNA molecule (DNA is synthesized from 5’ to 3’)
Okazaki
Fragment
Replication : 1 st step
• Unwind DNA
• helicase enzyme
• unwinds part of DNA helix
• stabilized by single-stranded binding proteins
single-stranded binding proteins
Replication : 2 nd Step
DNA
Polymerase III
▪ Build daughter DNA strand
◆
add new
complementary bases
◆
DNA polymerase III
But…
We’re missing something!
What?
Where’s the ENERGY
for the bonding?
Energy of Replication
energy
ATP GTP TTP CTP
Where does energy for bonding usually come from?
ADP AMP GMP TMP CMP
modified nucleotide
energy
We come with our own energy!
And we
leave behind a nucleotide!
rememberYou ATP!Are there other ways to get energy out of it?
Are there other energy nucleotides?
You bet!
Energy of Replication
• The nucleotides arrive as nucleosides
• DNA bases with P–P–P
• P-P-P = energy for bonding
• DNA bases arrive with their own energy source for bonding
• bonded by enzyme: DNA polymerase III
Replication
• Adding bases
• can only add nucleotides to
3 end of a growing DNA strand
• need a “starter” nucleotide to bond to
• strand only grows 5 → 3
DNA
Polymerase III DNA
Polymerase III
energy energy
3
3
5
The energy rules the process
5
Limits of DNA polymerase III
◆ can only build onto 3 end of an existing DNA strand
Leading & Lagging strands
5
5
5
5
3
3
3
5
3 5 3 3
Leading strand Lagging strand
ligase
Leading strand
◆ continuous synthesis
Lagging strand
◆ Okazaki fragments
◆ joined by ligase
▪ “spot welder” enzyme
DNA polymerase III
✓
3
5
growing replication fork
Formation of The Replication Fork
• The polymerase III holoenzyme binds to template DNA as part of multiprotein complex
• DNA polymerases only synthesize DNA in the 5’ to 3’ direction
• Because the DNA strand are antiparallel, the polymerase functions asymmetrically
• On the leading (forward) strand, the DNA is synthezied continuously
• On the lagging (retrograde) strand, the DNA is synthesized in
short (1-5 kb), the so-called Okazaki fragments
Replication fork
3’
5’
3’
5’
5’
3’
3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins primase
DNA
polymerase III DNA
polymerase III DNA
polymerase I
ligase
Okazaki fragments
leading strand
lagging strand
SSB
Formation of The Replication Bubbles
• Replication occurs in both directions along the length of DNA and both strand are replicated simultaneously
• This replication process generates “replication bubbles”
Replication Bubbles
DNA polymerase III
RNA primer
◆ built by primase
◆ serves as starter sequence for DNA polymerase III
Limits of DNA polymerase III
◆ can only build onto 3 end of an existing DNA strand
Starting DNA synthesis: RNA primers
5
5
5
3
3
3
5
3 5 3 5 3
growing
replication fork primase
RNA
DNA polymerase I
◆ removes sections of RNA primer and replaces with DNA nucleotides
But DNA polymerase I still can only build onto 3 end of an existing DNA strand
Replacing RNA primers with DNA
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
ligase
Telomeres
• In eukaryotic replication, following removal of RNA Primer from the 5’end of lagging strand : a gap is left
• This gap exposes DNA strand to attack of 5’
exonucleases
• This problem is overcome by Telomerase
Repeating, non-coding sequences at the end of chromosomes = protective cap
◆ limit to ~50 cell divisions
Telomerase
◆ enzyme extends telomeres
◆ can add DNA bases at 5 end
◆ different level of activity in different cells
▪ high in stem cells & cancers
telomerase
Telomeres
5
5
5
5
3
3
3
3
growing replication fork
TTAAGGG TTAAGGG
“Thank You”
