DNA Replication Flashcards
What is the direction of DNA synthesis
DNA is always synthesised in the 5’ to 3’ direction
What direction are the parental template strands “run”
The template strands are run in the 3’ to 5’ direction
What are the characteristics of eukaryotic DNA replication
DNA replication occurs from multiple origins of replications (ori)
It is bidirectional (replicating both ways from the origin)
What is meant by DNA replication being semi discontinuous
The leading strand is continuously sythensised in its 5’ to 3’ direction
The lagging strand is dis continuously synthesised in its 5’ to 3’ direction (as Okazaki fragments)
What is Primase
Primase is an enzyme that makes an RNA primer (a starting point for DNA polymerisation)
What is DNA polymerase III
An enzyme that synthesises a new DNA strand by adding nucleotides complementary to the parallel template strand
It only synthesises in the 5’ to 3’ direction
What is the “head of the zipper” where Helicase is unwinding DNA called
The replication fork
What is topoisomerase
An enzyme that cuts and rejoins DNA strands to release tension generated by the unwinding of DNA strands
What is DNA polymerase
An enzyme that detects DNA/RNA hybrids, degrades the RNA component, and fills the gap with DNA nucleotides
What is DNA ligase
An enzyme that joins the newly synthesised fragments (Okazaki fragments, ends of leading strands, gaps between replication bubbles)
Bonds created are phosphodiester bonds
What are the two activities that DNA polymerase 1 carries out
RNase activity - recognises DNA/RNA hybrid and degrades RNA component
DNA polymerisation activity - Synthesises DNA by adding nucleic bases in the remaining gaps
What provides progressive addition of DNA nucleotides
DNA polymerase 3
What provides a starting point for nucleotide addition
Primase enzyme which synthesises RNA primer
What unwinds helical double strand of DNA
Helicase
What releases tension built up by the unwinding of DNA helix
Topoisomerase
What prevents the unwound double strands from reforming, and protects them from degradation
Single stranded binding protein
What removes RNA primers, and fills the gaps with DNA nucleotides
DNA polymerase 1
What joins the ends of newly synthesised fragments together (Okazaki fragments, ends of leading strand, intersections of replication bubbles)
DNA ligase
When can DNA errors be repaired
During replication - through use of exonuclease
After replication - through use of endonuclease
Which enzyme has a DNA proof reading mechanism
DNA polymerase 3 has a preoofreading mechanism, where by it checks newly inserted nucleotide bases against the template
How are exonuclease errors removed
DNA polymerase 3 recognises damaged/incorrect DNA
Incorrect base is removed by the 3’ to 5’ exonuclease activity of DNA polymerase 3
DNA synthesis continues
What is the process of exonuclease
Damaged/incorrect DNA is identified during DNA polymerisation
Incorrect nucleotide is removed by the 3’ to 5’ exonuclease activity of DNA polymerase 3
DNA synthesis continues
What are examples of things than can cause DNA damage post replication
Incorrect inserted basses are not corrected by DNA polymerase 3
Radiation damage (e.g. UV light, falling inside Chernobyl)
Chemical modifications of bases (natural and chemical causes)
What is the process for endonuclease
Damage/incorrect DNA is located
Damage, including some flanking regions, is removed by an endonuclease
A DNA polymerase synthesises new DNA
DNA ligase joins new DNA to existing DNA (phosphodiester bonds)
What is the significance of a DNA error not being fixed
When the error carrying DNA helix is seperate for replication, both strands (including the error) will be used as a template. Hence the two parent strands will code the permanent DNA error onto the template strands and the error will be carried onto the two resulting new DNA strands. All future DNA molecules arising from this strand will carry this permanent error
What is “in vitro” DNA synthesis and PCR
DNA replication in a test tube
Polymerase Chain Reaction
What is the Polymerase Chain Reaction
“In Vitro” method of making multiple DNA copies so that there is enough DNA material to work with
Only “targeted” DNA region will be copied
Rapid exponential increase in DNA molecules
Method uses cycles of heating and cooling
What are some applications for the Polymerisation Chain Reaction
Medical applications
Forensic Applications
Infectious disease detection and identification
Molecular biology research applications
What are the 3 steps to polymerase chain reaction
Denaturation - Temperature is increased to seperate DNA strands
Annealing - Temperature is decreased to allow primers to base pair to complementary DNA template
Extension - Polymerase extends primer to form new (5’ to 3’) DNA strand
What occurs upon repetition of polymerase chain reaction
Exponential Amplification of the region of interest
What is the function of DNA template in PCR
DNA molecule to which complementary nucleotides can be matched to make identical copies via DNA synthesis
What is the function of primers in PCR
Defines region of DNA molecule to be replicated
Provides free 3’ OH group - essential to initiate DNA synthesis
What is the function of DNA polymerase in PCR
Enzyme which adds nucleotides (complimentary to DNA template) and joins them together using phosphodiester bond
What is the function of dNTPs in PCR
Free nucleotides - the building blocks used by DNA polymerase
