DNA Replication Flashcards
DNA polymerase III: role in DNA replication
- Most important protein involved in DNA replication
- Travels along the single DNA strand, adding the correct nucleotides to the free end of the new strand (3’ end).
- Also proofreads - after each nucleotide has been added, checks to make sure it is complementary to template base, if not, it’s excised and replaced.
Can DNA polymerase III start new DNA chains?
NO! Needs an RNA primer
Replication: How is RNA primer added to strand?
DNA polymerase III can’t start synthesizing on a bare strand, so….
Primase
- part of an aggregate of proteins called the Primeosome.
- This enzyme attaches a small RNA primer to the single-stranded DNA to act as a substitute 3’OH for DNA polymerase to begin synthesizing from (initation point of 3’ to 5’ parent chain)
- Attracts RNA nucleotides (“primers”) which bind to DNA nucleotides of 3’ to 5’ strand due to hydrogen bonds between bases
Replication: What are “primers”?
They are RNA nucleotides which bind to DNA nucleotides of 3’ to 5’ strand through hydrogen bonds between bases
DNA primase creates them - serve as starting point for DNA polymerase III.
How many strands act as a template
A single strand template, but both strands are used: S and S’
Where and why is deoxynucleoside triphosphate added?
to the 3’ end of the primer strand therefore chain growth is ALWAYS in the 5’ to 3’ direction
DNT loses two phosphates, becomes part of chain (nucleotide)
Antiparallel nature of DNA & DNA synthesis
Because the replication fork moves progressively, both strands of the DNA molecule must be synthesized at the same time. Since the DNA strands are antiparallel and DNA chain growth can only occur in the 5’ to 3’ direction, DNA synthesis on one strand must be discontinuous
Okazaki fragments
Lagging strand is 5’ to 3’, so the polymerase can’t move along it in continuous fashion.
As helicase “unzips” DNA, primase must continually start new primers in 3’ to 5’ direction (to cover exposed strand). After the primers, okazaki fragments can be laid down by DNA PIII.
How long are okazaki fragments?
1000-2000 nucleotides long in prokaryotes
100 to 200 nucleotides long in eukaryotes
First major step of DNA replication
The enzyme helicase breaks hydrogen bonds between bases of the two anti-parallel strands.
splitting happens where chains are rich in A-T (2 hydrogen bonds vs the three btwn Cytosine and Guanine).
- Name:
- initiation point where splitting starts
- structure that is created
initiation point where helicase splits the two strands: origin of replication
Resulting structure: Replication Fork
2 precursors to DNA polymerase copying DNA molecules
DNA molecules must be:
unwound by DNA helicases
stabilized by single stranded binding proteins (or would coil back)
(also primase?)
How do DNA helicases unwind duplex regions of DNA (and at what rate)?
rate of 1000 nucleotide pairs/second
Use ATP
Single-stranded binding proteins
Essential for DNA replication:
Stabilize unwound strands
Protect single-stranded DNA from attack by nucleases
SSBs and Hairpins
straighten unwound chain
if not, we’d have all kinds of coils and hairpins – but not enough cooperative binding to hold miles and miles of DNA straight (nor do we need to). But if it skips a part, it was not an accident
Hairpins are not random. Very very specific signals
Structure of Human SSB
RNA Primer Synthesis
- Primers for DNA polymerases are made by enzymes called DNA primases which use ribonucleotide triphosphates as substrates
- RNA follows the same base pairing rules as DNA thus the same templating rules are followed to create the RNA primer (except uracil)
- RNA primers are about 10 nucleotides long
- Lays w/3’HO end
What you see after okazaki fragments laid
Primers & Okazaki fragments. Some RNA in the chain
DNA Ligase
Adds phosphate to gaps in the lagging strand (sugar backbone)
Requires ATP!
Semi Conservative Replication
Daughter DNA duplex contains one parental strand and one newly synthesized strand
Replication origins
Where helicase binds in a double stranded DNA region to pry the two strands apart
In bacteria, yeast and many viruses, replication origins are specific DNA sequences adjacent to AT rich DNA regions that are easy to unwind
See E. coli example. takes 40 minutes to replicate 4.6x106 nucleotide pairs at the rate of 500-1000 nucleotides per second
How many forks at a time in DNA replication?
Many - not just one. And can be going in different directions. Which is leading and lagging depends on the fork, it seems.
