lecture 12 - DNA replication Flashcards
In what direction is DNA synthesised?
5’ to 3’ (5’ phosphate attaches to 3’ OH)
Where are the origins of replication in DNA replication?
At AT rich regions of DNA
Why are the origins of replication in AT rich regions?
Because A-T base pairs have 2 hydrogen bonds while G-C bonding is stronger because they have 3 hydrogen bonds.
What is the region where helicase continues splitting the strand, at the edges of replication bubbles?
Replication fork
What is a region of split DNA strands called?
Replication bubble
What is the leading strand in DNA replication?
The strand that, starting at an RNA primer, is continuously synthesised, from the 3’ to 5’ end of the template strand.
What is the lagging strand in DNA replication?
The side on the opposite side of the origin of replication, that forms Okazaki fragments (discontinuous synthesis)
What are the fragments made of DNA and RNA primer in the lagging strand of DNA replication?
Okazaki fragments
What are the chemicals/enzymes required for DNA replication?
helicase, primase, DNA polymerase III, topoisomerase, SSBP, DNA polymerase I, DNA ligase
How are the two DNA strands separated in semi-discontinuous DNA replication?
The enzyme helicase separates the strands at AT-rich regions
What is the role of primase in DNA replication?
Makes molecules that signal to enzyme that replicates DNA where to start along the strand.
Primase is an enzyme that makes RNA primers, which attach to the single DNA strand and lay down a section, leaving a 3’ OH group for DNA nucleotides to bond to, allowing the rest of the strand to form
What enzyme layers down RNA primers initially during DNA replication?
Primase
What is the role of DNA polymerase III in DNA replication?
Synthesised daughter strand DNA by adding nucleotides, complementary to the parental template strand.
How does DNA polymerase III start synthesising the DNA strand?
Starts at 3’ OH of RNA primer, then begins laying down nucleotides, with the bases complementary to the parent strand.
How is DNA synthesised in the lagging strand?
Okazaki fragments are formed when DNA polymerase III starts at 3’ OH of RNA primers at points all along the lagging strands, laying down DNA nucleotides
What is the process of laying down DNA nucleotides to form Okazaki Fragments?
Discontinuous synthesis
Why are Okazaki fragments seperate from each other/ why is the lagging strand discontinuous?
Because there are no phosphodiester bonds between the 5’ end of the RNA primers and the adjacent DNA nucleotides
What ‘end’ of a Okazaki fragment has the 5’ carbon group, and which has the 3’ carbon group?
RNA primer end has 5’, newly synthesised DNA has 3’ end
What does SSBP stand for, in terms of DNA replication?
Single strand binding proteins
What are single strand binding proteins?
Proteins that bind to single strands after they have been pulled apart by helicase, preventing them from bonding back together, or being degraded by other enzymes
What is the function of topoisomerase in DNA replication?
Nicks and rejoins DNA strands to release tension as the DNA uncoils and the replication bubbles expand.
What is the function of DNA polymerase I?
Uses endonuclease enzyme RNase H to recognise DNA:RNA hybrids (connections between RNA primers and synthesised DNA), and degrade the RNA, before DNA polymerase I synthesises DNA to replace these parts of the strand.
What is used to recognise DNA:RNA hybrids in Okazaki fragments, and remove RNA from the fragments?
the endonuclease enzyme RNase H
What is function of DNA ligase in DNA replication?
To join the fully synthesised Okazaki fragments together at 5’ and 3’ groups, removing the gaps and making the lagging strand continuous. Also joins leading and lagging strands, and strands from neighbouring replication bubbles
What is used to repair errors in DNA during replication?
Exonuclease
What is used to repair errors in DNA after replication?
Endonuclease
Why must errors in DNA base pairing be corrected?
If left un corrected, one half of the strand, when split, will replicate with the incorrect base and will then form DNA with an incorrect base pair. forming a permanent mutation
How does DNA pol III make repairs to DNA with errors during replication?
DNA pol III uses exonuclease to ‘proofread’ the newly inserted nucleotide, and compare to the the template/parent strand. If there is a fault (base attached to wrong base pair), the incorrect base is replaced.
How are errors in DNA formed after replication?
Radiation (UV), or chemical base modification
How does repair of DNA errors occur after replication?
Endonuclease removes the error and the flanking regions. DNA polymerase synthesises more DNA and then DNA ligase joins this new section to the existing DNA.
What is the name for in vitro DNA synthesis?
PCR (polymerase chain reaction)
What are the 3 key steps in PCR/in vitro DNA synthesis?
Denaturation, Annealing, Extension
What is the process of denaturation in PCR?
DNA is heated to high temperatures (94-98 degrees), which separates the strands
At what temperature range does denaturation occur in PCR?
94 to 98 degrees celsius
What is Annealing in PCR?
When the temperature of the DNA is decreased to 45-70 degrees, DNA primers will bind to complementary sections of DNA
What is the temperature at which Annealing in PCR occurs?
45 to 70 degrees
What is the process of extension in PCR?
DNA polymerase extends from DNA primers, synthesising DNA along the daughter strand.
The rate of replication occurring in PCR can best be described as?
Exponential Amplification
In PCR, does replication occur in continuous or discontinuous strands?
Two continuous strands
What are the components required for PCR?
Template DNA, DNA primers, DNA polymerase, dNTPS (deoxyribonucleic triphosphates - A,T,G,C)
What is required to bring in bases for replication during PCR?
dNTPs
What are dNTPs?
The free nucleotides that are added during PCR to act as the building blocks used by DNA polymerase to form the leading strand
