DNA Replication Machinery

Question 1

define semi-conservative DNA replication

Answer 1

each daughter double helix has a conserved parental/template strand and a newly synthesised/daughter strand

Question 2

what is required for chain elongation

Answer 2

free 3’ OH end
DNA polymerase
template strand
dNTPs
Mg2+

Question 3

what are Okazaki fragments
how long are they

Answer 3

regions of replicated DNA on the lagging strand created by the backstitching mechanism separated by primer gaps
100-200 bases long (EU)

Question 4

what determines the length of Okazaki fragments

Answer 4

length of space between nucleosomes

Question 5

number of replication origins in PRO vs EU chromosomes

Answer 5

PRO: one
EU: multiple, occur in clusters

Question 6

what is the role of initiator proteins/origin recognition complexes (ORC)

Answer 6

involved in assembly and loading of helicase at the origin

Question 7

In E. Coli what ensures DNA replication occurs only once per cell cycle

Answer 7

controlled accumulation of initiator protein DNAa at the origin

Question 8

what causes the refractory period after DNA replication (PRO)

Answer 8

initiator proteins bind to fully methylated regions
methylation occurs a short while after DNA replication

Question 9

explain how PRO initiator proteins cause DNA double helix to open

Answer 9

they wrap around it forming a DNA-protein filament that put torsional strain on the DNA which causes AT rich sequences to melt

Question 10

explain why (EU) ORC does not melt/unwind dsDNA

Answer 10

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

Question 11

what are the names of the replicative helicases in E. coli and EU

Answer 11

E. Coli: DNAb
EU: MCM complex (minichromosome maintenance protein complex)

Question 12

explain PRO E. Coli DNAb loading

Answer 12

• 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

Question 13

explain EU MCM helicase loading

Answer 13

• 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

Question 14

explain the role of single-stranded DNA binding proteins

Answer 14

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

Question 15

define priming

Answer 15

provides the 3’OH group paired to a DNA template required to initiate replication

Question 16

what enzyme synthesizes RNA primers

Answer 16

EU: DNA primase
PRO: primase (DNAg)

Question 17

which enzyme removes primers
which enzymes replaces RNA primers with DNA
which enzyme seals the nick in the backbone

Answer 17

primer removal: nuclease
primer replacement: polymerase
backbone nick sealing: ligase

Question 18

why use RNA primers and not DNA

Answer 18

they are automatically marked as ‘suspect copies’ and will always be removed and replaced with high fidelity

Question 19

why does it not matter that RNA primers have low fidelity (accuracy to template)

Answer 19

they will be removed and replaced by DNA with high fidelity

Question 20

explain exonuclease activity

Answer 20

in the case of a mismatch, the enzyme retreats 1 nt, polymerase activity ceases
the mismatched base is removed
enzyme makes a 2nd attempt

Question 21

role of each DNA polymerase in E. Coli

Answer 21

I: removes primer and replaces it
III: leading and lagging strand synthesis

Question 21

how does exonuclease distinguish which nucleotide is the daughter/parent strand

Answer 21

PRO: depends on methylation of A residues
EU: lagging strand gaps have clamps left by polymerase, direction faced by clamp orients repair machinery

Question 22

role of each polymerase in EU

Answer 22

alpha: removes primer and replaces it
epsilon: leading strand synthesis
delta: lagging strand synthesis

Question 23

what reaction does DNA ligase catalyse

Answer 23

formation of a phosphodiester bond between 3’OH and 5’P

Question 24

how is torsional stress of DNA relieved

Answer 24

DNA supercoiling
free rotation around a phosphodiester bond of a ss break

Question 25

what is the role of topoisomerases

Answer 25

produce transient ss or ds breaks in the backbone of DNA allowing rotation and re-ligation to prevent torsional strain

Question 26

how does DNA topoisomerase I work

Answer 26

covalently links to the backbone reversibly breaking the bond (via tyrosine) allowing rotation
reforms original bond so isoenergetic

Question 27

how does DNA topoisomerase II work

Answer 27

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

Question 28

why is a clamp required in DNA synthesis

Answer 28

on their own polymerases will synthesise a short string before falling off
releases polymerase when it reaches a ds region

Question 29

what are the names of the sliding clamp proteins in EU and PRO

Answer 29

EU: PCNA
PRO: polymerase III beta subunit

Question 30

explain the clamp loading mechanism

Answer 30

clamp loader (EU: RFC, PRO: gamma subunit complex)
clamp loader loads clamp onto DNA at primer junction by opening the ring

Question 31

why can the lagging strand final primer not be replaced

Answer 31

backstitching

Question 32

why must telomere be extended every cell division

Answer 32

prevents the loss of material with every primer gap by extending it

Question 33

explain telomerase activity

Answer 33

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)