L4 - DNA Replication Flashcards
What does the semi conservative nature of DNA replication mean?
Each of the parental strands acts as a template for the synthesis of the daughter strands
What direction can DNA rep occur in
5’ –> 3’
What is found at the 3’ end
Hydroxyl
What is found at the 5’ end
Phosphate
What can be said about the orientation of the template strands
They are antiparallel
Why is replication essentially irreversible
Because it is coupled to the breakdown of pyrophosphate making is highly unfavourable
What is the structure of pyrophosphate
2 inorganic phosphate molecules
What enzyme catalyses the breakdown of pyrophosphate
Pyrophosphatase
What is the equilibrium constant for the replication … what does this mean
10^5
Highly favoured in the forward direction
What is a dNTP
Dinucelotide tripohsophate
What is a dNMP
Dinucleotide monophosphate
What is the formation for replication
dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi ``
What enzyme seperates the paired DNA strands
DNA helicase
Why can the leading strand be continuously synthesied
5’ –> 3’ direction
What must the lagging strand be synthesied as
Okazaki framgments
All synthesis of DNA occurs as a result of extension of an
RNA primer
What enzyme lays the RNA primer
DNA primase
What does DNA primase require for it to work
DNA template
NTPs
What 4 enzymes are required for the synthesis of the lagging strand
DNA primase
DNA polymerase
Ribonuclease H
DNA ligase
Function of DNA primase
Makes and lays down the RNA primer
Function of DNA polymerase
Extension of the RNA primer with dNTP
Function of Ribonuclease H
Removes the RNA primer
What can be said about the specificity of ribonuclease H
Absolutely specific for RNA/DNA hybrid molecules
Function of DNA ligase
Seals the nick joining the Okazaki fragments
Does DNA ligase require ATP
Yes requires the energy of ATP hydrolysis
What is used in the first stage of ligation and released in the second
ATP used
AMP released
Describe the equations (two coupled) for ligation
ATP + 5’-P –> P-P + 5’-P-AMP
P-P –> 2 Pi + free energy
Describe the energy requirements of DNA helicase
Cont. using ATP
How does DNA used the energy released from ATP
Wraps around the parental strands when it spins the rotational force is converted to forward motion
ATP GIVES THE FORWARD MOMENTUM
Werners Syndrome
Progeria (premature ageing)
Autosomal recessive
In RECQ helicase gene WRU
Bloom syndrome
Rare cancer syndrome caused by loss of function in RecQ family DNA helicases which maintains the integrity of the genome
Describe how a sliding clamp is used to aid the processivity of DNA polymerase
Sliding clamp and clamp loader (w/ ATP) position close to primer;template interface
Clamp loader removed and replaced with DNA polymerase
The fact that DNA polymerase is unlikely to drop off can be summarised by what word
Processivity
What is the sliding clamp known as in humans
Proliferating cell nuclear antigen
What is the role of single strand binding proteins
Bind to the ssDNA of the replication fork making it available for synthesis and easing progression of the replication fork
Prevents any base pairing causing hair pins
SSBs bind cooperatively, what does this mean?
Once bound the probability of another one binding is high
What is the role of DNA topoisomerases - why are they needed
Prevent the DNA from becomming tangled
When the DNA is being unwound at the replication origin this introduces superhelical tension into the helix
role is to release the tension by nicking and resealing the backbone
TYPE 1 DNA TOPOISOMERASES
HOW DOES IT WORK
ATP?
Nick and reseal ONE of the two DNA strands
No atp demand
TYPE 2 DNA TOPOISOMERASES
HOW DOES IT WORK
ATP?
Nick and reseal both of the DNA strands
Requires ATP
Yeast replication origins
Autonomous replicating sequences (ARS) elements
Human replication origin
DNA sequence near to LMNB2 MYC HBB, but can also be defined by chromatin strucutre (lacking the nucelosomes)
Describe the importance of biphasic replication of the DNA
Temporal separation to ensure that each chromosome is replicated once per cycle
What is phase 1 of the biphasic replication
What cell cycle phase does it occur in
Replicator selection
G1
What is phase 2 of the biphasic replication
What cell cycle phase does it occur in
Origin activation
S
What occurs during replicator selection
Formation of the prereplicative complex
What occurs duing origin activation
Unwinding of the DNA and recruitment of DNA polymerases
Describe the phases of eukaryotic replicator selection
Origin recognition complex (ORC) binds
Helicase loading proteins, Cdc6 and Cdt1 bind to the ORC
Helicase Mcm2-7 binds to complete formation of the pre-RC
In G1 what can be said of the CDK levels
Low CDK
What is the effect of low CDK in G1
Allows formation of pre-RC complexes
Prevents activation of the pre-RC complex
In S phase what can be said of the CDK levels
High CDK
What is the effect of high CDK levels in S
Prevents formation of any pre-RC complexes
Allows activation of existing pre-RC complexes
What is the problem with finishing replication
Requirement of the RNA primer creates problems
Once ribonuclease H has removed all RNA primers, polymerase and ligase close all but one gap
Gives a natural tendancy of replicating chromosomes to become shorter
What is the effect of telomerase
Extends the 3’ end (overhang) to compensate for the natural shortening
What is the telomeric repeat
TTAGGG
Describe the strucutre of telomerase
Ribonucleoprotein
With an intrinsic RNA component which acts as template for the synthesis
What is the step wise telomere synthesis known as
The telomere shuffel
How many nucelotides does telomerase translocated
6 nucleotides
What is the 9 nucelotide RNA sequence on telomerase
AAUCCCAUU
What is meant by DNA replication being semi-conservative
Each new daughter strand produced in DNA replication consists of one parental helix and one newly synthesised DNA strand
What bonds are formed during the process of DNA replication
Phosphodiester bonds
An incoming nucleotide can only be added to the free 5’ hydroxyl group on the terminal deoxyribose sugar of an existing polynucleotide chain, T or F
F - incoming nucleotides can only be added to the free 3’ hydroxyl group
Recall the carbon nomenclature for a deoxyribose sugar
Carbon 1 (1’) is the carbon to the right of the oxygen atom in the deoxyribose ring. Move round in a clockwise direction so that carbon 5 (5’) is the CH2OH group attached to the deoxyribose ring
Describe the chemical mechanism for the addition of a new nucleotide to the growing polynucleotide chain
The 3’ deoxyribose sugar undergoes nucleophilic attack on the first phosphate bond of an incoming nucleotide trisphosphate
What is the other product formed from the addition of a nucleotide to the growing polynucleotide chain
Pyrophosphate
What attributes of DNA replication make it irreversible
The formation of a new phosphodiester bond and the production of pyrophosphate is coupled to a second reaction catalysed by pyrophosphatase that converts pyrophosphate to two molecules of inorganic phosphate (2Pi)
What reaction supplies the energy required for DNA synthesis
Breakage of 2 high energy phosphate bonds
State the general equation for the addition of a nucleotide to the grown polynucleotide chain
dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi
What chemical variables explain the irreversible nature of DNA synthesis
Equilibrium constant, Keq of the magnitude of 105 and the Gibb’s free energy, ?G = -7kcal mol-1
The irreversible nature of DNA synthesis accounts for the massive stability that is an inherent property of DNA molecules, T or F
T
What can be said about the orientation of the two polynucleotide chains in a dsDNA molecule
They are orientated antiparallel to each other
The primer sequence of DNA synthesis can only grow in a 3’ to 5’ direction, why is this
The addition of a dNTP can only occur by the nucleophilic attack of the 3’ carbon and hydroxyl on the 5’ phosphate of the incoming nucleotide
Due to the fact that DNA synthesis can only occur in a 5’–>3’ direction, how many polymerase enzymes are required per replication fork
2
Which enzyme is responsible for breaking the hydrogen bonds between complimentary base pairs
DNA helicase
What localised structure is formed by the breaking of hydrogen bonds between complimentary base pairs in the parent strand
Replication fork
Which enzyme is responsible for the synthesis of new DNA daughter strands
DNA polymerase
Which proteins maintain the unwound parental DNA strands in a single stranded conformation and hence ease replication fork progression
SSBs – single stranded binding proteins
DNA polymerase can only synthesis in a 3’ –> 5’ direction, T or F
F – vice versa
What is different between the synthesis of the leading and lagging strand during DNA replication
The leading strand is synthesised continuously and precedes the synthesis of the lagging strand. The lagging strand however, is synthesised discontinuously i.e. with breaks in the polynucleotide chain
What is the significance of DNA polymerases inability to work de novo
RNA primers required at the start of the replication of the leading strand and at the start of each Okazaki fragment of the lagging strand
What enzyme is responsible for the synthesis of RNA primers
DNA primase
RNA primers are required throughout both the leading and lagging strand synthesis, T or F
F – they are only required at the start of lagging strand synthesis but throughout leading strand synthesis
What two features are required for synthesis of RNA primers
DNA template strand and nucleotide trisphosphates
Explain the role of RNA primers in the synthesis of DNA
DNA polymerase requires an RNA primer in order to synthesise a new DNA strand. RNA primers supply DNA polymerase with base-paired chain ends to add new nucleotides to.
Explain how DNA polymerase synthesises the lagging strand
Lagging strand DNA polymerase completes Okazaki fragments in the 5’ to 3’ direction and then starts synthesising a completely new fragment further along towards the 5’ end of the parental template strand
What is the approximate size Eukaryotic primers and how often do they occur in the lagging strand
10 nucleotides long, occurring every 100-200 nucleotides in the lagging strand
What causes the synthesis of each Okazaki fragment in the lagging strand to halt
When DNA polymerase reaches the RNA primer attached to the 5’ end of another fragment
What is the initial product of DNA replication
A DNA-RNA hybrid molecule
Explain the role of ribonuclease H in DNA replication
Ribonuclease H removes the RNA primers in the initial DNA-RNA hybrid molecule
Which enzyme works with ribonuclease H to repair gaps in the DNA sequence
DNA polymerase – extends across the gaps created by RNA primer removal by ribonuclease H
Which enzyme joins 3’ and 5’ ends of the Okazaki fragments together
DNA ligase
Ligation of newly synthesised adjacent DNA fragments is a two-step reaction, requiring ATP hydrolysis, T or F
T
What attribute of the ligation of DNA fragments makes it another example of an irreversible reaction
Like DNA synthesis ligation results in the formation of another molecule of pyrophosphate. This reaction is then coupled to a reaction the converts PPi to 2Pi
Recall the general and word equation for the first reaction in DNA ligase activity
ATP + 5’P –> PPi + 5’P-AMP. Adenosine trisphosphate + 5’ phosphate –> pyrophosphate + 5’adenosine diphosphate
In the reaction catalysed by DNA ligase, two phosphates are removed from ATP to form AMP which then binds to the 5’ phosphate in the polynucleotide chain, the two phosphates are referred to as pyrophosphate, T or F
T
Recall the general and word equation for the reaction coupled to DNA ligase activity that accounts for its irreversible nature
PPi –> 2Pi + Free energy. Pyrophosphate is converted to two molecules of inorganic phosphate by pyrophosphatase, which also releases energy
The ligation process is rendered energetically highly favourable by the conversion of pyrophosphate (PPi) to 2Pi by pyrophosphatase, T or F
T
How does DNA helicase separate parental DNA strands at the replication fork
DNA Helicase sits like a nut on a bolt and uses the energy released by ATP hydrolysis to drive a rotational energy which is translated to a force that opens up the replication fork
Werner’s syndrome is an example of a disease caused by mutations in a helicase. Describe the aetiology and symptoms of this condition
Werner’s syndrome is a progeria (premature ageing) caused by a autosomal recessive mutation (loss of function) in the RECQ helicase encoded by the WRN gene
Give an example of another helicase mutation that causes disease, other than Werner’s syndrome
Bloom syndrome is another loss of function mutation that occurs in another Rec-Q family DNA helicase. The role of this helicase is to maintain genome integrity. This mutation causes a rare cancer phenotype with tumours in multiple tissues
What is meant by the processivity of DNA polymerases
Processivity refers the tendency of polymerases to continue to synthesise as long as there is sufficient substrate available
What protein is said to enhance the processivity of polymerases
Sliding clamp proteins
How do proteins that increase polymerase processivity act
Sliding clamp proteins keep the DNA polymerase enzyme at the primer template junction by fixing itself to the primer template junction through association with a protein called a clamp loader.
Describe the ternary structure formed by proteins that increase processivity and how this complex acts
Sliding clamp positioning is ATP-dependant. A ternary structure is formed by the sliding clamp and clamp loader proteins and the associated ATP. This complex sits behind the DNA polymerase and provides an extra impetus to drive it forward
What is significant about sliding clamp proteins across Eukaryotes
Extremely highly conserved
The human sliding clamp protein, proliferating cell nuclear antigen (PCNA) has a near identical structure to the homologue in yeast. What is PCNA significance in cancer
PCNA is a useful marker for hyperproliferative cells found in tumours
How do SSBs ease replication fork progression
They act to prevent hydrogen bond formation between complimentary base pairs within the same ssDNA strand by binding to the sugar-phosphate backbone and allowing easy progress of polymerase
What is the role of topoisomerases
Prevent the DNA from becoming tangled and supercoiled during DNA replication
How do topoisomerases act
Topoisomerases release the tension created in the polynucleotide chains created by unwinding of the two ssDNA strands. This is achieved by the selective nicking and resealing of regions in the DNA molecule
Explain the major differences between the two types of topoisomerases
Type I Topoisomerases nick and release one of the 2DNA strands and effective remove one turn in the molecule. This is an ATP-independent reaction. Type II Topoisomerases nick a reseal both DNA strands by causing dsDNA breaks effectively removing two turns of the supercoiled helix. This reaction is ATP-dependent
What is the name given to specific DNA sequences that direct the initiation of DNA replication by recruiting replication initiation proteins
Replicator sequences
Whilst in yeast, autonomously replicating sequences (ARS) direct DNA replication initiation, similar structures in humans have proven elusive but seem to be defined by chromatin structure rather than DNA sequence, T or F
T
Near to which genes have human DNA replication initiation sequences been found
LMNB2 (laminin B), MYC and HBB (Haemoglobin B)
Eukaryotic DNA replication is monophasic, T or F
F – its biphasic
Where in the cell cycle does replicator selection and formation of the pre-replicative complex occur
G1
What DNA replication event occurs in S phase
Origin activation – the unwinding of DNA and recruitment of DNA polymerase
Temporal separation of replicator selection and origin activation ensures what
Each origin is used and each chromosome is only replicated once per cell cycle
How does Eukaryotic replicator selection occur
Origin replication complex (ORC) binds to the replicator sequence. Helicase loading proteins then bind to ORC to convert the single stranded replicator sequence into a pair of replication forks. Mcm2-7 then also binds to complete formation of the pre-RC
Give example of helicase loading proteins that bind to ORC
Cdc6 and Cdt1
Cyclin dependent kinases are important in the temporal control of DNA replication. During which stage of the cell cycle are cdk levels particularly high and particularly low
G1 phase – low cdk activity. S phase – high cdk activity
How is cyclin-dependant kinase (cdk) activity important in limiting pre-RC formation and activation to specific points in the cell cycle
Cdk levels are high during S phase of the cell cycle. High cdk levels leads to the phosphorylation of already formed pre-RC thus activating them and leading to formation of the replication origin. Cdk also acts to phosphorylate the individual components of the pre-RC, particularly the Cdc6, Cdt1 and ORC elements. Phosphorylation of these constituent elements leads to their inactivation and hence inhibition of new pre-RC formation during S phase. During G1 cdk levels are low and thus there is little phosphorylation of Cdc6 and Cdt1 and hence more pre-RC formation
What accounts for the end replication problem during DNA replication
The need for an RNA primer for initiation of DNA synthesis. Ribonuclease H removes the last RNA primer in the linear chromosome sequence however DNA polymerase can no longer extend the DNA sequence. This would result in a gradual reduction in the length of replicated DNA by the length of one primer with each subsequent replication
How is the end replication problem overcome
Addition of non-coding telomeric repeat sequences to the 3’ end of the DNA sequence. These are long enough to enable DNA primase to bind and initiate new RNA primer synthesis and prevent chromosome shortening
Describe the composition of telomeric repeat sequences
Telomeres consist of a hexanucleotide repeat sequence (TTAGGG)
How does the telomerase enzyme act to create and maintain these telomeric repeats at the end of linear chromosomes
Telomerase is a ribonucleoprotein with an intrinsic RNA component containing the complimentary RNA sequence to the telomeric repeat sequence (AAUCCC). This RNA component acts as a template on which telomere repeat sequences are synthesised in a step-wise process known as the telomere shuffle. This allows addition of multiple TTAGGG repeats to the 3’-OH at each telomere.
