Lecture Notes

DNA synthesis & repair
Learning objectives:

1. How DNA is synthesised and replicated?

Possible essay from this lecture

Eukaryotic DNA polymerases

 Looking at genome replication during the S phase of the cell cycle
 Eukaryotes have at least 15 DNA polymerases
- Because there are many different type of DNA replication
 This lecture talk about genome replication, which includes:
- DNA polymerase
α
δ
ε
 other DNA polymerase ca replicate DNA but can also synthesis DNA or replicate mitochondrial DNA




Where is DNA replicated?

 Still don’t know
 Each eukaryotic chromosome contains many replicons
 Eukaryotic replicons are 40 to 100 kb in length
 Individual replicons are activated at characteristic times during S phase
 Regional activation patterns suggest that replicons near one another are activated the same time
 Initiation at oriC (is the beginning of the DNA replication site and also called origin ) requires the sequential assembly of a
large protein complex on the membrane
 DNA replication must be a highly controlled/ regulated process
- Because cancer cells have deregulated DNA replication hence rapid growth and division
- Increasing frequency of DNA replication = increase cell number
- In cancer, many mutations are targeted to the DNA replication machinery or more important the proteins that
regulate DNA replication

What Dr Hurd said

 There are certain regions in human genome where DNA replication begins

, There is a DNA sequence element that primes at the nuclear sites for DNA replication called origin
- Many origins have been characterised
 Regions where DNA replication originates are numerous and are spaced along chromosomes and result in more than 1
nucleation sites so that DNA polymerase proceeds away from the origin
 Ultimately get fusion of all the DNA bubble
 Causing the chromosome to separate and produce new genomes




What type of proteins regulate DNA replication?

 Initiation
- Creating the replication forks at the origin oriC
 Licensing factor
- Permit DNA replication to start
- A factor located in the nucleus and necessary for replication that is activated or destroyed after 1 round of replication
- New factors must be provided for further rounds of replication to occur
- Cell cycle regulated proteins
 DNA replication only occurs in the S phase of the cell cycle
- One way that cell controls DNA replication is by ensuring that the licensing factors are only present in the nucleus
during the start of DNA replication
- Many licencing factors are excluded from the nucleus in the new daughter cell or degraded after the S phase

, Proteins at the origin control susceptibility to initiation

 A number of protein factors are considered licensing factors, these include:
- MCM proteins
- Cdc6
- Cdt1
 The licensing factors permit the cell to enter the S phase
 The ORC (origin replication complex) is a protein complex that is associated with origins throughout the cell cycle
 Cdc6 protein is an unstable protein that is synthesised only in G1 (prior S phase)
 Cdc6 binds to the ORC and allows MCM proteins to bind
 Cdt1 facilitates MCM loading on origins
- MCM is a hexametric protein wrapped around the DNA, which means it is a helicase
- MCM are recruited to both ends of the DNA strand and start unwinding the DNA
- Cannot copy DNA unless unwound
 Once replication starts CDT1 and Cdc6 leave ORC