DNA Replication

Question 1

Function of dna replication

Answer 1

• Purpose is to copy all of the genetic material accurately before cell division so that both daughter cells receive a full complement of genetic material
• Replication errors cause mutations in the genetic code, which may be heritable if in germ line cells or somatic if in other cells e.g. cancer
• DNA replication occurs in S phase of cell cycle, tightly regulated
• Must only occur one per cell division cycle
• Daughter cells must only receive one genome copy
• Replication ‘starts’ at many points, needs co-ordination or you get chromosome instability and mis-expressed genes

Question 2

How do we know replication os semi conservative

Answer 2

meselson stahl
• Performed density gradient contrifugation to distinguish between E. coli duplex dna
• Parental dna labelled by growing bacteria in N15 for several generations
• Medium abruptly changed to contain only N14 and samples tested periodically
• First gen hybrid dna , second gen had one light band and one intermediate band
• Shows semi conservative replication

Question 3

Replication forks

Answer 3

• Replicating circular (bacterial) chromosomes appear as θ structures- known as replication eyes or bubbles
• Electron microscopy studies show two growing replication forms potentially moving away from a central origin
• Replication forks occur in AT rich regions as easier to pull apart dna here
• Replication forks are bidirectional
• To show bidirectionality bacillus subtilis cells were grown in presence of [H3]thymidine
• Weak emissions stop in photographic emulsion near point of origin
• Autoradiographs showed concentrated emissions at both forks

Question 4

Function of origin of replication

Answer 4

• DNA at replication origins contains short sequences that attract initiator proteins
• Bacterial chromosomes typically have a single origin of replication, 2 replication forks proceed in opposite directions until they meet halfway around the chromosome
• The only point where bacteria can contro dna replication is initiation
• Initiator proteins bind dna at replication origin to create dna-protein complexes
• Destabilises adjacent double helix
• Attracts 2 dna helicases, each bound to a helicase loader
• Loaders dissociate and helicase begins to unwind dna
• DNA primase synthesises first rna primers

Question 5

How is dna unwound

Answer 5

• DNA replication requires template strands
• Duplex dna is very stable
• Very high temperatures are required to separate the strands in vitro
• Solution: DNA helicase

Question 6

How to stop dna re-annealing

Answer 6

• DNA strands are spatially close and aligned
• Solution: Single-stranded DNA binding protein (SSB) in bacteria, RPA (replication protein A) in archea and eukaryotes

Question 7

How to unwrap dna

Answer 7

• DNA is a double helix whose strands wrap around each other once every 10.5 Bp
• In order for the replication fork to advance, the helix ahead would have to rotate rapidly
• This is damaging for the dna and can break dna
• Topological issue: circular dna or eukaryotic chromosomes, ends aren’t free to unwind, as the dna unwinds, positive super coils form ahead of the helicase
• As dna unwinds, superhelical turns will be introduced meaning the linking number (the number of times one strand crosses another) will remain the same
• This tightening of the helix will create intolerable strain and the energy required for unwinding the dna will become too great unless it is relaxed
• Solution: topoisomerases

Question 8

How to initiate chain synthesis

Answer 8

• DNA polymerase can’t initiate chain synthesis
• Can only add a nucleotide to the 3’ end of a base-paired nucleotide on the primer strand
• Solution: DNA primase

Question 9

DNA polymerase general

Answer 9

• Use single stranded dna as template
• Add dNTPs to free 3’-OH of a base paired nucleotide to synthesise a complementary strand
• Incoming nucleotides are selected by ability to form Watson-crick base pairs with template
• New dna strand forms duplex with template strand
• Synthesis is rapid, up to 1000 nucleotides per second
• Have proof reading activity to ensure accuracy
• Reaction driven by energy from the release of pyrophosphate and its subsequent hydrolysis to inorganic phosphate

Question 10

DNA polymerase III

Answer 10

• 5’-3’ polymerase activity
• 3’-5’ exonuclease activity (proof reading)
• Reverse direction, goes backwards and removes wrong nucleotide and replaces it with the right one

Question 11

DNA polymerase I

Answer 11

• 5’-3’ polymerase activity
• 3’-5’ exonuclease activity
• 5’-3’ exonuclease activity to remove rna primer

Question 12

DNA helicase

Answer 12

• A diverse group of enzymes
• Harness the hydrolysis of ATP
• Unwind short sections of AT-rich parental duplex DNA
• Specifically at recognised origins of replication
• Binds ssDNA and continues moving along the strand when it encounters dsDNA, thus prising the helix apart at a rate of 1000bp per second
• Same as processing of DNA polymerase III, no RDS at this stage
• DNA helicase melts dsDNA in vitro, powered by ATP hydrolysis

Question 13

SSB

Answer 13

• Coats single stranded DNA
• Must be stripped off for replication to occur
• Keep strands apart
• Stop the formation of secondary structures e.g. hairpins
• Help align strands
• Interact with other replication proteins at the replication forks
• Stimulate polymerases
• Each SSB protein prefers to bind next to a previously-bound protein
• This cooperative binding straightens out the dna template

Question 14

Topo Isomerase

Answer 14

• 2 classes:
• I makes ss breaks, e.g. if tension in molecule
• II makes staggered ds breaks e.g. used in mitosis
• Cause transient interruptions of dna backbone then reseal them
• Mechanism:
• One end of dna helix can’t rotate relative to the other end
• Type I DNA topoisomerases with tyrosine at active site
• DNA topoisomerase covalently attaches to a dna phosphate, breaking a phosphodiester linkage in one dna strand
• The two ends of the dna double helix can now rotate relative to each other, relieving accumulated strain
• The original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible
• Spontaneous re-formation of the phosphodiester bond regenerates both the dna helix and the dna topoisomerase

Question 15

DNA primase

Answer 15

• Catalyses synthesis of a short rna primer at the origin of replication, then stops
• Unlike dna polymerase it can start a new polynucleotide chain by joining 2 ribonucleotides together
• DNA polymerase can then catalyse the addition of deoxynucleotides to the 3’ end
• DNA helicase and dna primase together comprise the primosome

Question 16

How does cell ensure dna is only copied once

Answer 16

• Many replication forks along each chromosome simultaneously
• Different cells use different sets of origins
• May allow a cell to coordinate its active origins with other features of its chromosomes e.g. which genes are being expressed
• Replication forks are formed in pairs and move in opposite directions away from a common point of origin, stopping when they collide with another replication bubble so that two complete daughter dna helices are formed
• Initiation of eukaryotic replication is regulated to ensure that dna is only replicated once
• In G1 phase the replicative helicases are loaded onto dna next to orc to create a prereplicative complex.
• Upon passage from G1 phase to s phase, specialised protein kinases activate the helicases, the unwinding of the dna allows loading of remaining replication proteins e.g. dna pol
• Protein kinases that trigger dna replication simultaneously prevent assembly of new prereplicative complexes until next m phase resets cycle
• They do this by phosphorylating orc so that it can’t accept new helicases
• Because of this each origin of replication can only fire once during each cell cycle

Question 17

Why is replication semi-discontinuous

Answer 17

• DNA pol can’t work from 3’ to 5’ so both strands can’t be synthesised continuously
• Leading strand synthesis is continuous in direction of replication fork movement
• Lagging strand is also synthesised in 5’-3’ but discontinuously as a series of short dna pieces called Okazaki fragments
• The synthesis of each Okazaki fragment ends when dna pol runs into a new rna primer attached to the 5’ end of a previous fragment
• These are joined by dna ligase to make long dna chains
• Elongation of the lagging strand is in the opposite direction to the direction of replication fork advance

Question 18

Priming semi discontinuous replication

Answer 18

• While leading strand synthesis requires one rna primer, lagging strand requires multiple primers, one for each fragment
• These are synthesised by dna primase

Question 19

Origins of replication

Answer 19

• From a single origin (OriC, AT-rich in e.coli) these 2 forks advance in opposite directions at constant speed and meet up half way around the chromosome
• OriC is bound by an initiator protein DnaA that opens up a 45bp segment into single strands. DnaC binds and permits helicase, DnaB, binding
• Helicase unwinds DNA powered by ATP hydrolysis
• Replication terminates at specific Ter sites
• At the end of replication, the two double-stranded daughter chromosomes are interlocked and need to be separated by a type II DNA topoisomerase
• There are thousands of origins of replication forks in a eukaryotic cell
• A eukaryotic chromosome typically contains 60 times more dna than a prokaryotic chromosome

Question 20

Processivity of dna pol I and III

Answer 20

• DNA pol I is used to remove RNA as it has low processivity so dissociates from DNA
• DNA pol III has high processivity

Question 21

DNA pol III structure

Answer 21

• DNA pol III is a holoenzyme
• Has a sliding clamp to keep dna pol in contact with strand
• Has proof reading 3’-5’exonuclease
• Beta clamp subunit is a sliding clamp
• There is also a clamp loader than can slide left to right to stop polymerase sliding off dna
• This increases processivity
• Core has polymerase activity
• If an enzyme carries out a single reaction and then dissociates from substrates = distributive
• If an enzyme performs multiple actions before dissociating = processive
• DNA pol can dissociate easily from the template, allowing it to recycle and begin synthesis of the next Okazaki fraction
• The sliding clamp keeps the dna polymerase on the dna while it is moving and increases the enzymes processivity
• When the polymerase encounters a ds region the clamp releases it
• Assembly of clamp requires atp hydrolysis by clamp loader at a primer-template junction

Question 22

Accuracy of dna replication

Answer 22

• Erasable rna primers are used instead of dna as any enzyme that starts chains can’t be efficient at self-correction so if the primers were retained in the genome there would be a high error rate
• Reliance on complementary base pairing is not sufficient to ensure accuracy
• Proof-reading mechanisms ensure that fidelity is very high- only 1 error for every 10^9 nucleotides copied

Question 23

2 mechanisms for ensuring accuracy of replication

Answer 23

• 1st mechanism:
• Correct nucleotide has higher affinity for polymerase as it correctly base-pairs with template
• Phosphodiester bond formation involves conformational change in DNA polymerase so incorrectly-bound nucleotides do not fit active site
• 2nd mechanism:
• 3’-5’ exonuclease activity of dna polymerase
• Takes place immediately after incorrect addition to growing chain
• Template strand contains methylated residues, new strand doesn’t contain methylated residues yet so new dna strands can be transiently distinguished from old ones so the enzyme knows which base to correct ( in bacteria)
• In eukaryotes, the newly synthesised lagging strand contains nicks before they are sealed by dna ligase
• Polymerase can’t extend such a strand- requires a base-paired 3’-oh end of the primer strand
• Clips off mismatches at the separate catalytic site
• The newly synthesised dna transiently unpairs and the polymerase undergoes a conformational change, moving the editing catalytic site into place for removal
• Mismatched base removed and re synthesis of the excised segment, using old strand as template

Question 24

Replisome

Answer 24

• Replication complex:
• Most enzymes held together in large, multi enzyme replication complex
• At front of fork dna helicase opens double helix
• 2 dna pol molecules work at the fork, one leading one lagging
• Made possible by folding back of lagging strand
• DNA primase also linked

Question 25

What would happen if replication on either side was independent

Answer 25

• Pol III’s could end up 3kb away from each other
• DNA helicase could generate excess lagging strand template
• So:
• Lagging strand template dna loops round, bringing thr 2 dna polymerases into a complex
• This brings the 3’ end of a completed Okazaki fragment close to the start site for the next fragment
• Known as the trombone model
• strands are synthesised simultaneously, although lagging strand synthesis begins a little after than the other

Question 26

What is the RDS of replication

Answer 26

• Unwinding is Rate limiting step
• DNA unwinding and synthesis is concomitant
• After sufficient ssDNA is produced, lagging strand synthesis can occur
• Discontinuous due to Okazaki fragment creation and rna primer synthesis
• As lagging strand template is exposed it binds ssDNA binding protein
• Each fragment requires a new rna primer

Question 27

Lagging strand synthesis

Answer 27

• After completing a fragment the lagging strand holoenzyme relocates to a new primer
• locked in with beta clamp
• When the polymerase encounters the previously synthesised fragment, the pol III core releases the DNA and loses its affinity for the beta clamp
• It is held in place by connections to the pol III core involved in leading strand synthesis

Question 28

Completion of lagging strand synthesis

Answer 28

• DNA pol III elongates chain from primer then falls off
• DNA pol I binds and used 5’-3’ exonuclease acitivity to remove the rna from the Okazaki fragment, replacing it with dna using 5’-3’ polymerase activity
• DNA ligase links Okazaki fragments in a reaction requiring ATP in eukaryotes or NAD in e.coli

Question 29

Applications of dna replication enzymes to recombinant dna technology

Answer 29

• DNA ligase can be used in recombinant dna technology
• Taq polymerase used in PCR
• Polymerase used in Sanger sequencing