Lecture 6 Flashcards
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
T
Where in the cell cycle does replicator selection and formation of the pre-replicative complex occur
G1
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
