TMC5 Flashcards
What is the difference between G1, G0, S, G2 and M phases of the cell cycle?
G0 - Non-dividing state
G1 - Cell grows and prepares for cell division, checks previous cycle was completed correctly and cell cycle checkpoints, cell arrests if checkpoints aren’t met
S - DNA replication phase
G2 - Cell prepares for mitosis by growing, producing proteins, increased organelles
M - mitosis and cell division
What is an origin of replication?
The points on DNA where replication begins
What is the difference between NTPs and dNTPs?
NTPs - precursors of RNA
dNTPs - precursors of DNA
List the items required by DNA polymerase for DNA synthesis
dNTPs
Primers
Template strand
What are the differences between RNA polymerase and DNA polymerase?
RNA polymerase:
De novo
Produces a single strand
DNA:
Not de novo
Produces a double strand
What is a primer in DNA synthesis?
The start point of DNA synthesis by DNA polymerase, synthesises from the 3’ end of an existing piece of RNA/DNA (primer)
Explain how DNA polymerase synthesises a new strand of DNA.
Uses one strand of DNA as a template
Synthesises DNA onto the 3’ end of a primer
Attaches a dNTP onto the 3’ OH group of a deoxyribose (5’ to 3’)
Extends a primer annealed to a template
Catalyses the formation of a phosphodiester bond
High energy dNTP is consumed in the reaction releasing pyrophosphate
What is the direction of DNA synthesis?
5’ to 3’
Describe the properties of DNA polymerase.
Purified DNA polymerase can replicate DNA in vitro
Error rate is 1 in 100,000
Proofreading carried out by a 3’-5’ exonuclease activity
Needs a primer
What is the basic DNA polymerase error rate?
1 in 100,000
Describe proof reading by DNA polymerase.
Decreases error rate in DNA synthesis by 100 fold
Carried out by a 3’ to 5’ exonuclease activity in DNA polymerase (digests DNA 3’ and moves toward 5’)
Nucleotide misincorporated during DNA replication
Immediately slows down polymerisation by DNA pol by 10,000 fold
Increases the 3’ to 5’ exonuclease activity
Describe mismatch repair
About 1% of the time, a misincorporated nucleotide is not excised by proofreading and remains in DNA creating a mismatch base pair
Corrects mismatched base pair
Mismatch repair system must be able to discriminate between the old strand of DNA and the newly synthesised DNA so that it can remove the mismatched base from the new strand
Explain the differences between DNA polymerase proof reading and mismatch repair.
Proofreading by the DNA polymerase during DNA synthesis reduces error by about 100 fold.
Mismatch repair (MMR) repair occurs after DNA synthesis and reduces error by another 100 fold.
What is HNPCC and what are the characteristics of HNPCC?
Hereditary Nonpolyposis Colorectal Cancer
Defects in mismatch repair system
Cancer of colon and rectum
Increased risk of cancers of GI tract and endometrium
3 relatives over 2 generations with colorectal cancer, 2 must be first degree relatives, 1 must be under 50, FAP must be excluded
What is the genetic basis of HNPCC?
Mutations in any of the mismatch repair genes for the proteins that recognise different forms of mismatched base pairs in DNA cause HNPCC
MLH1, MSH2, MSH6, MSH3, PMS1, PMS2
Explain how DNA replication is initiated.
Recognition:
Complex of proteins binds the origin of replication - key one being DnaA
ORC binds to ori in eukaryotes
Melting:
Proteins at ori recruit proteins and melts DNA at ori
Unwinding:
Helicases unwind DNA and cause a replication bubble at ori
Single stranded binding protein binds to the single stranded DNA and keep it single stranded so that replication can proceed
Recruitment:
Proteins assembled at ori must recruit DNA polymerase
Primer synthesis:
RNA primer is synthesised at ori by DNA primase
Supercoiling builds up at edges of opening
Supercoiling relieved by topoisomerases I and II in humans and DNA gyrase in prokaryotes
DNA polymerase then starts synthesising
List the proteins involved in initiating DNA synthesis and each of their functions.
DnaA binds at ori
ORC binds at ori in eukaryotes
ORC recruited proteins melt DNA
Helicases unwind DNA and cause replication at bubble
Single stranded binding protein binds to the single stranded DNA and keep it single stranded so that replication can proceed
RNA primer is synthesised by DNA primase
Topoisomerase relieves supercoiling
DNA gyrase relieves supercoiling in prokaryotes
DNA polymerase synthesises DNA
Explain leading and lagging strand DNA synthesis.
Leading strand is the DNA synthesised from the RNA primer that can continue all the way to the end of the DNA molecule
Two replication forks one travelling right and the other left
DNA replication occurs on both template strands at the same time in the same direction
5’-3’ action on one of the strands is achieved by Okasaki fragments on one strand being joined up to allow overall progress in a 3’-5’ direction
Okasaki fragments are started from a RNA primer
Sealed by DNA ligase after DNA polymerase removes primers
DNA pol I binds to 3’ of Okaskai fragment and extends fragment by synthesising new DNA til it meets a primer
DNA pol I has 5’ to 3’ exonuclease activity
DNA pol I then dissociates after primer is degraded leaving a nick which is sealed by DNA ligase
In humans, primer is removed by Fen-1 and RNAseH and the gap is closed by DNA polymerase
What is the function of DNA polymerase I in E. Coli?
Proofreading, primer removal
What is the function of DNA polymerase III in E. Coli?
Lagging strand synthesis
Leading strand synthesis
Proofreading
What is the function of DNA helicase in E. Coli?
Unwinds DNA from helix
What is the function of DNA ligase in E. Coli?
Seals nicks in DNA
What is the function of DNA primase in E. Coli
Primer synthesis
What is the function of DNA topoisomerase?
Relieves supercoiling in front of replication fork
