DNA Replication Machinery Flashcards
define semi-conservative DNA replication
each daughter double helix has a conserved parental/template strand and a newly synthesised/daughter strand
what is required for chain elongation
free 3’ OH end
DNA polymerase
template strand
dNTPs
Mg2+
what are Okazaki fragments
how long are they
regions of replicated DNA on the lagging strand created by the backstitching mechanism separated by primer gaps
100-200 bases long (EU)
what determines the length of Okazaki fragments
length of space between nucleosomes
number of replication origins in PRO vs EU chromosomes
PRO: one
EU: multiple, occur in clusters
what is the role of initiator proteins/origin recognition complexes (ORC)
involved in assembly and loading of helicase at the origin
In E. Coli what ensures DNA replication occurs only once per cell cycle
controlled accumulation of initiator protein DNAa at the origin
what causes the refractory period after DNA replication (PRO)
initiator proteins bind to fully methylated regions
methylation occurs a short while after DNA replication
explain how PRO initiator proteins cause DNA double helix to open
they wrap around it forming a DNA-protein filament that put torsional strain on the DNA which causes AT rich sequences to melt
explain why (EU) ORC does not melt/unwind dsDNA
ORC is involved in loading the helicase in an inactive state during G1
activation occurs in S phase
this ensures replication occurs once per cell cycle
what are the names of the replicative helicases in E. coli and EU
E. Coli: DNAb
EU: MCM complex (minichromosome maintenance protein complex)
explain PRO E. Coli DNAb loading
- helicase loader DNAc binding to DNAb opens the hexameric ring
- ssDNA enters through the crack and binds the central channel
- DNAb ring closes and is active
- DNAc releases and leaves
explain EU MCM helicase loading
- open ring of ORC binds Cdc6 and this encircles DNA
- this complex and Cdt1 load an open ring hexameric MCM complex
- Cdc6 and Cdt1 are released upon MCM binding
explain the role of single-stranded DNA binding proteins
assist in DNA helix opening by stabilizing unwound ss conformation and prevent hydrolysis and hairpin formation
facilitate transfer of 3’ OH end of primers or DNA between polymerases
define priming
provides the 3’OH group paired to a DNA template required to initiate replication
what enzyme synthesizes RNA primers
EU: DNA primase
PRO: primase (DNAg)
which enzyme removes primers
which enzymes replaces RNA primers with DNA
which enzyme seals the nick in the backbone
primer removal: nuclease
primer replacement: polymerase
backbone nick sealing: ligase
why use RNA primers and not DNA
they are automatically marked as ‘suspect copies’ and will always be removed and replaced with high fidelity
why does it not matter that RNA primers have low fidelity (accuracy to template)
they will be removed and replaced by DNA with high fidelity
explain exonuclease activity
in the case of a mismatch, the enzyme retreats 1 nt, polymerase activity ceases
the mismatched base is removed
enzyme makes a 2nd attempt
role of each DNA polymerase in E. Coli
I: removes primer and replaces it
III: leading and lagging strand synthesis
how does exonuclease distinguish which nucleotide is the daughter/parent strand
PRO: depends on methylation of A residues
EU: lagging strand gaps have clamps left by polymerase, direction faced by clamp orients repair machinery
role of each polymerase in EU
alpha: removes primer and replaces it
epsilon: leading strand synthesis
delta: lagging strand synthesis
what reaction does DNA ligase catalyse
formation of a phosphodiester bond between 3’OH and 5’P
how is torsional stress of DNA relieved
DNA supercoiling
free rotation around a phosphodiester bond of a ss break
what is the role of topoisomerases
produce transient ss or ds breaks in the backbone of DNA allowing rotation and re-ligation to prevent torsional strain
how does DNA topoisomerase I work
covalently links to the backbone reversibly breaking the bond (via tyrosine) allowing rotation
reforms original bond so isoenergetic
how does DNA topoisomerase II work
covalently links to both strands of the DNA at the same time, activated by sites where two helices cross over each other (like in supercoiling). 1) breaks open on double helix to create a DNA “gate”. 2) causes the second double helix to pass through the opening. 3) reseals the break
why is a clamp required in DNA synthesis
on their own polymerases will synthesise a short string before falling off
releases polymerase when it reaches a ds region
what are the names of the sliding clamp proteins in EU and PRO
EU: PCNA
PRO: polymerase III beta subunit
explain the clamp loading mechanism
clamp loader (EU: RFC, PRO: gamma subunit complex)
clamp loader loads clamp onto DNA at primer junction by opening the ring
why can the lagging strand final primer not be replaced
backstitching
why must telomere be extended every cell division
prevents the loss of material with every primer gap by extending it
explain telomerase activity
binds with an RNA template that is part of the complex to the template DNA and extends the 3’ end allowing primase to add a primer and polymerase to complete the lagging strand (except the new primer gap)
