DNA replication Flashcards
Helicase function
unwinds double stranded DNA by breaking H bonds between bases, forming single stranded DNA
whats required to add nucleotides
a template strand (complementary bases)
an enzyme (DNA pol) and its cofactor (Mg2+)
a substrate (dNTP)
a 3’ hydroxyl group to start adding bases from (RNA primer)
adding RNA primer
DNA pol-α contains a primase anneals RNA primer to the template strand
Leading strand synthesis
DNA pol-ε adds bases from 3’ hydroxyl group of primer from 5’ to 3’ end, adds bases continuously in the direction towards the replication fork
Lagging strand synthesis
DNA pol-δ is unable to add bases from 3’ to 5’ direction, adds bases in short discontinuous pieces called okazaki fragments
RNA primer is required to start each okazaki fragments
how are RNA primers removed and gaps filled in
RNAseH proteins cleave primers, DNA polymerase fills in spaces left behind with nucleotides
role of DNA ligase
joins fragments together with phosphodiester bonds
other proteins involved
PCNA (sliding clamp proteins) - allows stable binding of DNA pol, hence efficient strand synthesis
SSBP/RPA (single stranded binding proteins) - stabilises single stranded DNA so it doesnt reanneal before replication
DNA Gyrase - form of topiomerase, relieves torsional stress, prevents DNA overtwisting
actual chemical reaction between DNA pol and dNTP
3 aspartic acid residues in active site, 2 Mg2+ ions, negatively charged phosphate groups
Mg interacts with both Asp and phosphate, enzyme cleaves 2 phosphate groups from dNTP, joining with phosphodiester bond to 3’ hydroxyl group
cleaving is energetically favourable
semi conservative
each progeny cell contains 1 DNA strand from parent and 1 DNA strand that is newly synthesised, meaning both strands are copied in DNA replication
high fidelity of DNA pol
δ and ε have 3’ to 5’ exonuclease activity, able to detect misalignment of 3’ hydroxyl when wrong nucleotide is added, goes back to correct the mistake
low error rate
how are histones added to newly synthesised DNA
DNA must be packed simultaneously with histones
CAF-1 delivers histones to replication fork to form nucleosomes
some histones from original parents chromosome and some are newly synthesised (random process)
purpose of histone tail
can be modified for epigenetics
DNA replication direction and origins
both directions with multiple origins
3 elements for chromosome to be copied
centromere (constricted region)
replication origins
telomeres (G rich tandem repeated sequences)
replicon definition
amount of DNA replicated within 1 origin
timing and order of chromosome replication
during S phase
regions of chromosome are replicated synchronously, others replicated at distinct times
heterochromatin replicates late, euchromatin replicates earlier
ORC
origin replication complex, binds to origins and separates DNA strands by recruiting replication proteins
licensing origins
ensures inititation is once per origin and only once per cell cycle
