Lecture 1: DNA Replication - Problems and Players Flashcards
Why is DNA replicated?
The purpose is to copy ALL of the genetic material ACCURATELY before cell division so that both the daughter cells receive a full complement of genetic material.
Why is it so important that DNA replication is so accurate?
Replication errors cause mutations in the genetic code, which may be heritable if in germ-line cells or somatic if in other cells (e.g. cancer).
Discuss the regulation and coordination of the eukaryotic cell cycle.
- In the mammalian cell cycle, DNA replication occurs during ‘S’ phase (synthesis phase), preceding mitosis (M), and is tightly regulated.
- DNA replication must occur only once per cell division cycle. Daughter cells must receive only one genome copy.
- Multiple parts of the genome are replicating at the same time. This needs to be highly regulated so all chromosomes are replicated only once (not missing or multiple)
- Replication “starts” at many points: needs co-ordination. Otherwise you get chromosome instability & mis-expressed genes
Describe the 3 possible DNA replication scenarios.
1) semi-conservative: one parental strand and one new strand per double helix. This is the correct scenario.
2) conservative: two parental strands stay together and the daughter duplex is completely new
3) dispersive replication: old DNA becomes fragmented and disperses with the new
Describe Meselson and Stahl’s experiment which proved one of the DNA replication scenarios.
In 1958, Meselson & Stahl performed a CsCL equilibrium density-gradient centrifugation to distinguish between E. coli duplex DNA containing only 14N only 15N or a mixture of both.
- Parental DNA labelled by growing bacteria in 15N for several generations – H/H duplex only (two heavy 15N strands)
- Medium abruptly changed to contain only 14N and samples tested periodically.
Expected outcomes:
- If ‘Conservative’ theory is correct: the 2 “heavy” parental strands remain together. Daughter duplex has two “light” strands. Both will separate from each other on gradient
- If ‘Semi-Conservative’ theory correct: after 1 generation, the 2 “heavy” parental strands separate & will bind to a “light” partner creating a hybrid with a density halfway between H/H parental & L/L ordinary
- If ‘Dispersive’ theory is correct: Strands would have a mix of 14N & 15N so would have appeared as an intermediate density
After one generation we cannot tell the difference between semi-conservative or dispersive theories (both have one band between the light and heavy positions).
Actual Results:
After the second generation there is the band midway between light and heavy and a band at heavy.
This proves the SEMI-CONSERVATIVE theory.
What can be seen when the circular DNA of a prokaryote undergoes replication?
A replication eye or bubble, which appear as theta structures. At both ends of the replication eye is a replication fork, each of which is moving away from a central origin. Replication forks permit genomic stability during replication as the amount of single stranded DNA is kept to a minimum (single stranded DNA can be damaged by enzymes & mechanical shear).
Are replication forks unidirectional or bidirectional?
Bidirectional. This means that there is replication at both of the two replication forks in the replication eye.
How was bidirectionality of the replication forks shown?
To show “bidirectionality”:
- Bacillus subtilis cells were grown in the presence of [3H]thymidine.
- Weak emissions stop in photographic emulsion near point of origin.
- Autoradiographs showed concentrated emissions at both forks.
What are DNA polymerases?
DNA polymerases are the enzymes used in prokaryotes and eukaryotes to replicate DNA.
Some characteristics:
- They use single-stranded DNA as a template
- They add dNTPs to free 3’-OH of a base-paired nucleotide to synthesise a complementary strand
- Incoming nucleotides are selected by ability to form Watson-Crick base pairs with template
- New DNA strand forms duplex with template strand.
- Synthesis is rapid (up to 1000 nucleotides per second)
- Have proof-reading activity to ensure accuracy
- Have 2 Mg^2+ (magnesium) ions: one bends/binds to the free OH’, allowing the nucleophilic attack
Which reaction does a DNA polymerase catalyse?
Polymerases catalyse addition of a dNTP (deoxynucleotide) to the 3’-OH end of a polynucleotide chain, a reaction driven by energy from the release of pyrophosphate (PPi) and its subsequent hydrolysis to inorganic phosphate, PPi –> 2 Pi (releases a lot of energy and makes the reaction energetically favourable as a whole).
What are the names of the two main DNA polymerases?
DNA polymerase III and DNA polymerase I
Which different activities do the two main DNA polymerases have?
DNA polymerase III:
5’ – 3’ polymerase activity
3’ – 5’ exonuclease activity (proof-reading)
DNA polymerase I: 5’ – 3’ polymerase activity 3’ – 5’ exonuclease activity (proof-reading) AND 5’ – 3’ exonuclease activity
Both have 5’ – 3’ polymerase activity and 3’ – 5’ exonuclease activity (proof-reading), but only DNA polymerase I has 5’ – 3’ exonuclease activity.
What is the function of DNA helicases in DNA replication?
DNA helicases unwind the double stranded DNA to allow the strands to be used as templates in replication. These enzymes are required because the DNA duplex is very stable and would require very high temperatures to denature the DNA into single strands without an enzyme.
Helicases
- use energy from the hydrolysis of ATP to unwind DNA
- unwind short sections of AT-rich duplex DNA
- unwind DNA specifically at recognised origins of replication
- has to initially bind to single stranded DNA
In prokaryotes - run along the lagging strand
In eukaryotes/archaea - run along the leading strand
How are the separated DNA prevented from reannealing?
The two separated DNA strands are very ‘sticky’, because they are spatially close and aligned for correct base pairing.
Single-stranded DNA binding proteins (called SSB in bacteria and RPA (replication protein a) in archaea and eukaryotes) coat the separated single-stranded DNA and prevents the strands from reannealing. These proteins must be stripped off before replication can continue along that stretch of template DNA.
Each SSB protein prefers to bind next to a previously-bound protein. This cooperative binding straightens out the DNA template. If the DNA template is not straight, DNA polymerase cannot run along it.
Single-stranded DNA binding proteins (SSBs):
- are homotetramers
- Keep strands apart
- Stop the formation of secondary structures (e.g. hairpins)
- Help align strands
- Interact with other replication proteins at the replication forks
- Stimulate polymerases
- protects the unwound DNA from nuclophiles/ROS/enzymes in the cell, which it would otherwise be vulnerable to when unwound, by looping the DNA over itself
What are the functions of topoisomerases in DNA replication?
Short answer: Topoisomerases cause transient breaks in the DNA backbone to relieve tension and prevent excessive supercoiling ahead of the DNA helicase, and then reseal the backbone after DNA helicase has passed.
Long answer:
Topological issue: Circular DNA or eukaryotic chromosome ends aren’t free to unwind, so as the DNA unwinds, positive supercoils form ahead of the helicase.
As DNA unwinds superhelical turns will be introduced meaning the linking number (the no. of times one strand crosses another) will remain the same.
This tightening of the helix will create intolerable strain and the energy required for unwinding the DNA will become too great unless it is relaxed.
Cairns , 1963, suggested there must be some form of “swivel” to allow rotation of the strands. If there was a “nick” in one of the strand’s phosphodiester bond the other could swivel round.
This nick is introduced by an enzyme called a topoisomerase. There are two types of topoisomerase:
- Topoisomerase I: makes single-stranded breaks
- Topoisomerase II: makes staggered double-stranded breaks.
The topoisomerases cause transient breaks in the DNA backbone to relieve tension and then reseal the backbone after DNA helicase has passed.
