lecture 4 - 07/10/24 Flashcards
Who discovered DNA replication and what is it?
Meselson and Stahl
1958
half old DNA, half new DNA
Is DNA replicated before or after cell division?
before
S -phase
Why was cesium chloride used in the 1958 discovery of semiconservative replication?
It has a similar weight as DNA
Describe the semiconservative experiment
- 2 samples of bacteria grown in different N-
containing (N-14 and N-15) mediums - isolate N-DNA and load into centrifuge tube
- centrifuge at high speed for 48hrs to form a cesium
chloride density gradient - N-14 lighter so closer to top of the tube
- N-15 heavier so closer to bottom of the tube
- as gen number increases, [N-14] increases and [N-
15] decreases in population
What is needed in DNA synthesis?
- DNA polymerase + Mg2+ (helps maintain the
mixture) - dNTPs
- single stranded template DNA
- primer 3’ -OH
- 5’ –> 3’ direction
Describe DNA synthesis events
- complementary base pairing
- 5’ –> 3’ direction
- requires 3’ -OH residue to extend from
- breakage of phosphoanhydride bond (P-O bond
between phosphate groups) of dNTP - formation of phosphodiester bond
What are telomeres?
Areas of highly repetitive DNA that protect chromosome ends from DNA degradation, recombination, and end fusion with other chromosomes
What are centromeres?
repetitive DNA which forms the spindle attachment site in mitosis
What is the origin of replication?
special sequence where duplication of the DNA begins; each chromosome will have many origins
Describe the movement of direction of DNA replication from the origins
bidirectional
Eukaryotic genomes vs prokaryotic genomes
eukaryotic
large
linear chromosomes
prokaryotic
(usually) circular
compact
small
How many chromosomes are in humans?
23 pairs of chromosomes
How many origins of replication does bacterial DNA have?
1
replication forks
- leading strand synthesis is continuous
- lagging strand synthesis is discontinuous (okasaki
fragments are joined together) - on lagging strand DNA polymerase ‘jumps’ to next
section and reads short segments 5’ –> 3’
describe lagging strand synthesis
- new RNA primer synthesised by primase
- DNA polymerase adds nucleotides to 3’ end of
new RNA primer to synthesis okazaki fragment - previous RNA primer removed by nucleases and
replaced with DNA by repair polymerase - nick sealed by DNA ligase
What enzymes proteins work at the replication fork and what do they do?
enzymes act together as a REPLICATION MACHINE
Primase - synthesise RNA primer
sliding clamp - keeps hold of DNA strand and keeps DNA polymerase on DNA as it is in a free flowing cytoplasm
DNA helicase - unwind DNA
single stranded DNA-binding proteins - stabilise DNA
DNA polymerases - synthesis new DNA strands
What do topoisomerases do?
it untwists the supercoiled DNA (caused by helicase unwinding DNA) ahead of the replication fork by breaking and reforming phosphodiester bonds
What is MutS?
- Mismatch repair protein
- scans along DNA looking for DNA kinks caused by
mismatched base pairs and recruits DNA repair
proteins to them to put in the correct base - mutations in human Mismatch Repair genes are
associated with predisposition to some cancers
replication errors
5’ –> 3’ polymerisation 1 in 10^5
(errors per nucleotide added)
3’ –> 5’ exonucleolytic proofreading 1 in 10^2
(errors not corrected)
strand-directed mismatch repair 1 in 10^3
(errors not corrected)
combined 1 in 10^10
Summary of DNA replication
- helicases unwind the parental double helix
- single-stranded binding proteins stabilize the
unwound parental DNA - leading strand is synthesised continuously 5’ –>
3’ by DNA polymerase - lagging strand synthesised discontinuously.
primase synthesises a short RNA primer, which is
extended by DNA polymerase to form an okasaki
fragment - after the RNA primer is replaced by DNA, DNA
ligase joins the okasaki fragment to the growing
strand
