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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)


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


What two features are required for synthesis of RNA primers

DNA template strand and nucleotide trisphosphates


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


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

DNA ligase


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


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


Explain the role of ribonuclease H in DNA replication

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


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

dNTP + (dNMP)n --> (dNMP)n+1 + 2Pi


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


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


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


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


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


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


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

They are orientated antiparallel to each other


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


What bonds are formed during the process of DNA replication

Phosphodiester bonds


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



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



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.


What is significant about sliding clamp proteins across Eukaryotes

Extremely highly conserved


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


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


What is the initial product of DNA replication

A DNA-RNA hybrid molecule


What DNA replication event occurs in S phase

Origin activation – the unwinding of DNA and recruitment of 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


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


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