Week 4 Flashcards
ingredients for DNA synthesis
- origin of replication
- primers
- dNTPs
- ATP as an energy source
- DNA polymerase
- accessory proteins
DNA synthesis procedure
- separate DNA strands
- synthesise DNA
- proofread newly synthesised DNA
bacterial DNA replication
- origin of replication attracts initiator proteins, which bind to the origin of replication sequence.
- unwinding by a helicase
- binding of single-strand DNA binding proteins (ssps) which keeps replication fork open
- RNA primers made by primase
- DNA polymerase makes the new DNA strand
- sliding clamp holds polymerase onto DNA
- nick sealing by DNA ligase (getting rid of RNA primers, filling the gap, and sealing gaps between Okazaki fragments)
initiator proteins for replication in e.coli
- binds to origin - very specific and regulate when DNA replication happens.
- destabilise the helix
- helps helicase bind, acting as a signpost for DNA helicase to bind to helicase-loading proteins
- requires ATP. don’t hydrolyse it until DNA synthesis has begun because of regulation. until new ATP is achieved a new cycle of DNA replication cannot be started
describe how helicase works
- two types of helices exist
- predominant one moves 5’ to 3’ along the lagging strand template
- helicases require ATP
- quaternary structure
single-stranded binding proteins
- following the action of helicase, single strand binding proteins keep DNA strands separated by binding ssDNA, which has short regions of base-paired ‘hairpins’
- single-strand binding protein monomers undergo cooperative protein binding, which straightens the region of the chain
RNA primers made by primase
- primase (a type of RNA polymerase) used to make RNA primers
- in order to begin, DNA polymerase requires a bound primer
- primase proceeds in 3’ to 5’ along template strand, primase synthesis occurs in the 5’ to 3’
- primase joins together two ribonucleotides and continues
- associated with Helicase
dna polymerase
- DNA is made in a complementary and antiparallel way using base pairing
- nucleotides are added onto the 3’ OH
- incoming nucleoside triphosphate pairs with a base in the template strand
- DNA polymerase catalyses covalent linkage of nucleoside triphosphate onto growing new strand
sliding clamp
circular protein
- does not impede progress of DNA polymerase
how are Okazaki fragments on lagging strand linked together?
- DNA polymerase adds nucleotides to 3’ end of new RNA primer to synthesise Okazaki fragment
- DNA polymerase finishes Okazaki fragment
- previous RNA primer removed by nucleases and replaced with DNA by repair polymerase
- tiny gap in the phosphodiester bond, nick, (no bases missing) sealed by DNA ligase
how are nicks healed?
ATP hydrolysed, AMP released
bacterial replisome
example of a molecular machine responsible for DNA replication in bacteria
bacterial primosome
DNA helicase and primase
what is the unwinding problem and how is it solved?
- as helicase unwinds DNA, supercoiling and tosional strain increase in the absence of topoisomerase, as DNA cannot rapidly rotate
- problem in circular chromosomes and large linear eukaryotic chromosomes
- some torsional stress is released by DNA supercoiling
- DNA topoisomerase creates a transient single-stranded break
- torsional stress ahead of the helicase is relieved by free rotation of DNA around the phosphodiester bond opposite the single-strand break; the same DNA topoisomerase that produced the break reseals it
what happens at the ends of linear chromosomes? (eukaryotic problem only)
- no 3’ OH group available at the end of the chromosome on the lagging strand
- lagging strand is incompletely replicated
- the potential shortening of the 5’ end of the daughter DNA is a problem as it may lead to a loss of sequence information
telomerase
- contains an RNA template which determines the repetitive sequence that is added to the 3’ end of the parental strand
- able to add DNA nucleotides to the 3’ end of parental DNA in the absence of a primer or DNA template
- RNA template -> DNA comp. copy
what enzyme does telomerase resemble?
reverse transcriptase but has an RNA template
what is generated by telomere replication?
G-rich end
telomeres and cancer
- telomerase is abundant in stem and germ-line cells, but not in somatic cells
- loss of telomeres, which occurs normally during DNA replication limits the number of rounds of cell division
- most cancer cells produce high levels of telomerase
3-20 kb of (TTAGGG)
reduced kb of (TTAGGG)
ends with no kb of (TTAGGG)
up to 55kb of kb of (TTAGGG)
embryonic or stem cell - indefinite replication
somatic cell - limited replication
senescent cell - breakage-fusion-bridge-cycle, chromosome instability, apoptotic cell death
cancer cell - persistent growth, but also chromosome instability - breakage and deletions
does solving the end replication problem require an RNA primer?
yes
RNA/DNA polymerases have an error rate of
1 in 10^4; 1 in 10^9
the human genome (3x10^9) in a haploid cell is only changed about — nucleotides every time a cell divides
3
2 separate mechanisms of DNA proofreading and repair
- 3’ to 5’ exonuclease - removes the disincorporated nucleotide, but only from the ends. DNA polymerase has proofreading exonuclease activity. flips the newly synthesised strand from the polymerising domain into the editing domain (the 2 domains of DNA polymerase), removing the base from 3’ to 5’.
- strand-directed mismatch repair - this is a DNA replication error repair process (if proofreading fails). initiated by detection of distortion in the geometry of the double helix generated by mismatched base pairs.
Describe strand-directed mismatch repair in detail
- MutS protein recognises and locks onto DNA mismatch.
- MutS recruits MutL and scans DNA.
- sliding clamp is encountered. MutL nuclease is activated and initiates strand removal
- strand removal completed
- repair DNA synthesis by polymerase
methylation in prokaryotes
MutL detects which strand is methylated to determine which is the original (correct) strand and which is the newly synthesized (possibly incorrect) strand.
defects in repair mechanisms
- linked to a variety of human diseases
- eg breast, colon, skin cancers
DNA can be damaged by:
- oxidation
- radiation
- heat
- chemicals
thymine dimer
UV radiation may cause: one base is covalently bonded to the other; this causes a problem because when DNA polymerase is trying to replicate DNA, because these bases are stuck to each other, the polymerase will skip over the dimer and keep on going.
C-C and C-T dimers may also occur
depurination
the base can be chopped off of the DNA strand for guanine and adenine
deamination
cytosine becomes uracil as NH2 is replaced by O
two general mechanisms of DNA repair long after DNA replication is over
- base excision repair
- nucleotide excision repair
base excision repair
repairs one nucleotide
- Uracil DNA glycosylase looks for U’s (deaminated C’s) that don’t belong in the DNA helix
- once it finds it, it will remove the base
- AP endonuclease and phosphodiesterase remove the remaining sugar phosphate
- DNA polymerase adds new nucleotide using bottom strand as a template
- DNA ligase seals the break
nucleotide excision repair
larger types of repairs (‘bulky’ lesions)
- eg pyrimidine dimers or remove benzylpyrine in smokers
- an excision nuclease will excise a big chunk of DNA from where the problem is
- DNA helicase is also needed to open things up to remove the fragment
- DNA polymerase adds new nucleotides using bottom strand as a template
- DNA ligase seals break
non-homologous end joining
- there is an accidental double-strand break
- processing of DNA end by nuclease
- end joining by DNA ligase
- the break is thus repaired with some loss of nucleotides at the repair site
homologous recombination
- there is an accidental double-stranded break
- there is processing of the broken ends by recombination-specific nuclease
- the double-strand break is accurately repaired using undamaged DNA as the template
- the break is thus repaired with no loss of nucleotides at the repair site
when does DNA polymerase undergo a small structural rearrangement that allows it to catalyse the nucleotide-addition reaction?
only when the base-pairing between each incoming nucleoside triphosphate and the template strand is correct
clamp loader
uses the energy of ATP hydrolysis to lock the sliding clamp onto DNA
