DNA Synthesis and the cell cycle Flashcards
Why is DNA replication described as semi-conservative?
Because half of each of the daughter Strands produced are made up of parental DNA while the other half is made up of newly synthesised DNA.
At what point on the DNA is DNA replication started?
DNA replication is initiated at specific sites on the DNA called replication origins
Why is Eukaryotic DNA replication initiated at multiple points on the chromosomes?
Because there’s to much DNA to copy meaning DNA replication would take longer than the time it takes for a whole mammalian cell to replicate
What is the origin recognition complex and what does it do?
The origin recognition complex is a 6-subunit protein. It’s role is to recognise and then bind to the replication origins on the DNA
What are the 6 subunits that make up the origin recognition complex?
ORC1, ORC2, ORC3, ORC4, ORC5 and ORC6
What structure forms once the ORC binds to the replication origins and how does it form?
pre-replication complex is formed. It is formed when all the other proteins required for DNA replication, e.g. Cdc6 (cell division cycle 6), dock onto the ORC to form the large complex.
What happens once the pre-replication complex is formed?
Once the pre-replication complex is formed the DNA unwinds to form a replication bubble
What stage of the mammalian cell cycle does the pre-replication complex form?
Late M phase/G1 phase
How long does the bacterial cell cycle last?
20-30 mins
What are the stages that make up the bacterial cell cycle?
S phase and M phase. (S phase takes up majority of cell cycle)
How long does the mammalian cell cycle last?
16-24 hours
What are the stages that make up the mammalian cell cycle? what occurs during each phase?
M phase - Cell division
G1 phase - Synthesis of histones and DNA polymerase (preparation for S phase)
S phase - DNA synthesis
G2 phase - Preparation for M phase
What is DNA polymerase?
DNA polymerase is the enzyme that synthesises new DNA strands from parental DNA
How many DNA polymerases are present within the human genome?
15
Examples of Eukaryotic DNA polymerases
Alpha - DNA replication Beta - DNA repair Gamma - Mitochondrial DNA replication Delta - DNA replication Epsilon - DNA replication
Examples of Prokaryotic DNA polymerase
I - DNA repair
II - DNA repair
III - DNA replication
Key properties of DNA polymerase
- always acts in the 5’ to 3’ direction (adds nucleotides, deoxyribonucleoside triphosphates, to the 3’ OH end of the newly synthesised DNA strand)
- requires a DNA template strand; a DNA or RNA primer; the 4 deoxyribonucleoside triphosphates, dNTP’s, and Mg2+ ions in order to synthesise new DNA
- utilises the A-T and C-G base pairs to synthesise a new DNA strand
- has proof-reading/editing functions
What are the 4 deoxyribonucleoside triphosphates?
The 4 dNTP’s are: dATP, dCTP, dGTP and dTTP
Why is a primer required for DNA replication?
DNA or RNA primer required as DNA polymerase unable to synthesise new DNA completely from scratch
Describe the reaction that allows DNA polymerase to elongate the new DNA strand that it synthesises?
- nucleotide complementary to the nucleotide present on the parental strand binds to active site of DNA polymerase
- Lone pair of electrons of OH- ion present on 3’OH end of previous nucleotide attack delta positive alpha - phosphate of next nucleotide
- This causes a phosphodiester bond to form between OH- and the alpha - phosphate thus linking the previous and new nucleotides together
- This also cause the release of pyrophosphate from the new nucleotide (provides energy for reaction to occur).
- DNA polymerase contains inherent pyrophosphatase enzyme which brakes down pyrophosphatase preventing reaction from being reversed.
What is the mechanism that occurs to allow elongation of the new DNA strand to occur?
Nucleophilic substitution
What are the 2 strands produced at a replication fork called?
leading strand and lagging strand
Why can the leading strand be made continuously?
Because the leading strand is being made in the 5’ to 3’ direction which is moving in the same direction as the replication fork.
Why must the lagging strand be made discontinuously?
Because the strand is being made in the 5’ to 3’ direction but is moving away from the replication fork.
How is the synthesis of the lagging strand different to the synthesis of the leading strand?
Leading strand only requires one DNA primer and so is synthesised in one long continuous strand.
The lagging strand however, requires many different RNA primers to be added to it in order for the DNA polymerase to continue elongating it. This means the lagging strand is synthesised in fragments.
What are the name of the fragments produced as a result of the discontinuous synthesis of the lagging strand?
Okazaki fragments
What is the role of Primase/Alpha DNA polymerase in DNA replication?
produces RNA primers and adds them to 5’ end in the 5’ to 3’ direction to the leading and lagging strand of the replication fork. Primase does this in prokaryotic cells but in Eukaryotic cells alpha DNA polymerase does this.
What role does DNA helicase have in DNA replication?
uses ATP to expose the parental DNA strands by breaking hydrogen bonds between the bases thus causing the DNA to unwind and form the replication fork
What role do the single stranded binding proteins have in DNA replication?
prevent single stranded parental strands from annealing back to another
What role does repair DNA polymerase have in DNA replication?
It replaces the RNA primers used to direct the DNA polymerase producing the lagging strand with DNA
What role does DNA ligase have in DNA replication?
forms phosphodiester bonds between the DNA bases that replaced the RNA within the primer
What role do gamma and epsilon DNA polymerase have in DNA polymerase?
DNA polymerase binds to primer and elongates it (sliding clamp holds the DNA polymerase in place)
DNA polymerase then translocates across the parental strand and catalyses the formation of the phosphodiester bonds between the nucleotides within the newly synthesised leading strand.
Controls what nucleotides form part of new strand by only allowing the complementary nucleotide into its active site.
What role does the enzyme exonuclease have in DNA polymerase?
Once leading strand fully formed RNA in primers removed by an exonuclease and replaced by DNA
What role do DNA Topoisomerase I and II play in DNA replication?
DNA Topoisomerase I goes ahead of the DNA helicase and removes both positive and negative supercoils (works by cleaving one strand of DNA and then resealing it once positive or negative supercoil is removed). DNA Topoisomerase II functions same way but uses ATP and can cleave both strands of DNA).
What is the error rate of DNA polymerase?
1 in 10^8 base pairs
Why is the error rate so low for DNA polymerase?
Due to the complementary base pairing and due to the proof-reading/editing function of the DNA polymerase
What is the error rate of DNA replication overall?
1 in 10^10 base pairs
Why is the error rate of DNA replication so low?
Mismatch repair system- corrects most of the polymerase errors. Multi-enzyme system highly conserved across species
