Lecture 6

Question 1

What attributes of DNA replication make it irreversible

Answer 1

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)

Question 2

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

Answer 2

F - incoming nucleotides can only be added to the free 3’ hydroxyl group

Question 3

What two features are required for synthesis of RNA primers

Answer 3

DNA template strand and nucleotide trisphosphates

Question 4

Explain how DNA polymerase synthesises the lagging strand

Answer 4

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

Question 5

Which enzyme joins 3’ and 5’ ends of the Okazaki fragments together

Answer 5

DNA ligase

Question 6

How is cyclin-dependant kinase (cdk) activity important in limiting pre-RC formation and activation to specific points in the cell cycle

Answer 6

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

Question 7

RNA primers are required throughout both the leading and lagging strand synthesis, T or F

Answer 7

F – they are only required at the start of lagging strand synthesis but throughout leading strand synthesis

Question 8

Explain the role of ribonuclease H in DNA replication

Answer 8

Ribonuclease H removes the RNA primers in the initial DNA-RNA hybrid molecule

Question 9

State the general equation for the addition of a nucleotide to the grown polynucleotide chain

Answer 9

dNTP + (dNMP)n –> (dNMP)n+1 + 2Pi

Question 10

How do topoisomerases act

Answer 10

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

Question 11

How does DNA helicase separate parental DNA strands at the replication fork

Answer 11

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

Question 12

Give an example of another helicase mutation that causes disease, other than Werner’s syndrome

Answer 12

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

Question 13

Describe the ternary structure formed by proteins that increase processivity and how this complex acts

Answer 13

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

Question 14

Which enzyme works with ribonuclease H to repair gaps in the DNA sequence

Answer 14

DNA polymerase – extends across the gaps created by RNA primer removal by ribonuclease H

Question 15

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

Answer 15

G1 phase – low cdk activity. S phase – high cdk activity

Question 16

What is different between the synthesis of the leading and lagging strand during DNA replication

Answer 16

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

Question 17

What can be said about the orientation of the two polynucleotide chains in a dsDNA molecule

Answer 17

They are orientated antiparallel to each other

Question 18

What attribute of the ligation of DNA fragments makes it another example of an irreversible reaction

Answer 18

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

Question 19

What bonds are formed during the process of DNA replication

Answer 19

Phosphodiester bonds

Question 20

Ligation of newly synthesised adjacent DNA fragments is a two-step reaction, requiring ATP hydrolysis, T or F

Answer 20

T

Question 21

Where in the cell cycle does replicator selection and formation of the pre-replicative complex occur

Answer 21

G1

Question 22

How do proteins that increase polymerase processivity act

Answer 22

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.

Question 23

What is significant about sliding clamp proteins across Eukaryotes

Answer 23

Extremely highly conserved

Question 24

Recall the carbon nomenclature for a deoxyribose sugar

Answer 24

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

Question 25

Werner’s syndrome is an example of a disease caused by mutations in a helicase. Describe the aetiology and symptoms of this condition

Answer 25

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

Question 26

What is the initial product of DNA replication

Answer 26

A DNA-RNA hybrid molecule

Question 27

What DNA replication event occurs in S phase

Answer 27

Origin activation – the unwinding of DNA and recruitment of DNA polymerase

Question 28

Which proteins maintain the unwound parental DNA strands in a single stranded conformation and hence ease replication fork progression

Answer 28

SSBs – single stranded binding proteins

Question 29

How do SSBs ease replication fork progression

Answer 29

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

Question 30

Due to the fact that DNA synthesis can only occur in a 5’-->3’ direction, how many polymerase enzymes are required per replication fork

Answer 30

2

Question 31

Describe the composition of telomeric repeat sequences

Answer 31

Telomeres consist of a hexanucleotide repeat sequence (TTAGGG)

Question 32

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

Answer 32

T

Question 33

Eukaryotic DNA replication is monophasic, T or F

Answer 33

F – its biphasic

Question 34

What causes the synthesis of each Okazaki fragment in the lagging strand to halt

Answer 34

When DNA polymerase reaches the RNA primer attached to the 5’ end of another fragment

Question 35

What is the role of topoisomerases

Answer 35

Prevent the DNA from becoming tangled and supercoiled during DNA replication

Question 36

Give example of helicase loading proteins that bind to ORC

Answer 36

Cdc6 and Cdt1

Question 37

Near to which genes have human DNA replication initiation sequences been found

Answer 37

LMNB2 (laminin B), MYC and HBB (Haemoglobin B)

Question 38

How does the telomerase enzyme act to create and maintain these telomeric repeats at the end of linear chromosomes

Answer 38

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.

Question 39

What is the approximate size Eukaryotic primers and how often do they occur in the lagging strand

Answer 39

10 nucleotides long, occurring every 100-200 nucleotides in the lagging strand

Question 40

What accounts for the end replication problem during DNA replication

Answer 40

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

Question 41

Which enzyme is responsible for breaking the hydrogen bonds between complimentary base pairs

Answer 41

DNA helicase

Question 42

Recall the general and word equation for the reaction coupled to DNA ligase activity that accounts for its irreversible nature

Answer 42

PPi --> 2Pi + Free energy. Pyrophosphate is converted to two molecules of inorganic phosphate by pyrophosphatase, which also releases energy

Question 43

How does Eukaryotic replicator selection occur

Answer 43

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

Question 44

The primer sequence of DNA synthesis can only grow in a 3’ to 5’ direction, why is this

Answer 44

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

Question 45

What reaction supplies the energy required for DNA synthesis

Answer 45

Breakage of 2 high energy phosphate bonds

Question 46

What localised structure is formed by the breaking of hydrogen bonds between complimentary base pairs in the parent strand

Answer 46

Replication fork

Question 47

What is meant by DNA replication being semi-conservative

Answer 47

Each new daughter strand produced in DNA replication consists of one parental helix and one newly synthesised DNA strand

Question 48

How is the end replication problem overcome

Answer 48

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

Question 49

Recall the general and word equation for the first reaction in DNA ligase activity

Answer 49

ATP + 5’P --> PPi + 5’P-AMP. Adenosine trisphosphate + 5’ phosphate --> pyrophosphate + 5’adenosine diphosphate

Question 50

What enzyme is responsible for the synthesis of RNA primers

Answer 50

DNA primase

Question 51

What is meant by the processivity of DNA polymerases

Answer 51

Processivity refers the tendency of polymerases to continue to synthesise as long as there is sufficient substrate available

Question 52

What is the name given to specific DNA sequences that direct the initiation of DNA replication by recruiting replication initiation proteins

Answer 52

Replicator sequences

Question 53

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

Answer 53

PCNA is a useful marker for hyperproliferative cells found in tumours

Question 54

What is the significance of DNA polymerases inability to work de novo

Answer 54

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

Question 55

The irreversible nature of DNA synthesis accounts for the massive stability that is an inherent property of DNA molecules, T or F

Answer 55

T

Question 56

Explain the major differences between the two types of topoisomerases

Answer 56

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

Question 57

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

Answer 57

T

Question 58

The ligation process is rendered energetically highly favourable by the conversion of pyrophosphate (PPi) to 2Pi by pyrophosphatase, T or F

Answer 58

T

Question 59

DNA polymerase can only synthesis in a 3’ --> 5’ direction, T or F

Answer 59

F – vice versa

Question 60

Temporal separation of replicator selection and origin activation ensures what

Answer 60

Each origin is used and each chromosome is only replicated once per cell cycle

Question 61

Explain the role of RNA primers in the synthesis of DNA

Answer 61

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.

Question 62

Which enzyme is responsible for the synthesis of new DNA daughter strands

Answer 62

DNA polymerase

Question 63

What protein is said to enhance the processivity of polymerases

Answer 63

Sliding clamp proteins

Question 64

Describe the chemical mechanism for the addition of a new nucleotide to the growing polynucleotide chain

Answer 64

The 3’ deoxyribose sugar undergoes nucleophilic attack on the first phosphate bond of an incoming nucleotide trisphosphate

Question 65

What is the other product formed from the addition of a nucleotide to the growing polynucleotide chain

Answer 65

Pyrophosphate

Question 66

What chemical variables explain the irreversible nature of DNA synthesis

Answer 66

Equilibrium constant, Keq of the magnitude of 105 and the Gibb’s free energy, ?G = -7kcal mol-1