Average Process
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what are the 3 stages of DNA replication
Initiation
Elongation
termination
define the initiation stage of DNA replication
loading of replication proteins
define the elongation stage of replicatio
copying of the dna
define the termination stage of DNA replication
removing of the replicaiton proteins
what is the product of DNA replication?
2 sister chromatids
what is the function of topoisomerase enzymes?
resolves the supercoils of DNA
what does helicase do
seperates the strands to give single stranded DNA
what does DNA ligase do?
seals DNA ends together
what is mean by "not all origins of replication are fired at the same time"
some areas of the genome are copied before others
what is the product of not all areas of the genome being replicated at the same time?
early and late synthesis stages of the genome
what happens during G1 phase to the DNA replication machinery?
the G1 replication proteins are recruited to the replication origins
- e.g DNA helicase loaded onto the DNA
what process activates the replicaiton proteins during the G1 - S phase transitio
phosphorylation of the proteins
how many primers does the leading and the lagging strand need?
leading only needs the 1 but the lagging strand needs many due to discontinous replication
what DNA polymerase is responsible for the leading strand and which is responsible for the lagging strand?
leading is polymerase epsilon
lagging is polymerase delta
what causes termination of replication
TOP2 cleavage of DNA structures + ubiquitination of proteins
what are the 2 forms of replication stress?
DNA damage during DNA replication
when replication forks slow down
give some intracellular obsticles which can introduce replication stress
:)
transcription here = transcription conflict
give an example of DNA secondary structures and what they do?
G quadruplets which impede movement of DNA polymerase
what are RNA loops as intracellualr obsticles?
DNA - RNA hybrids which impeed the movement of replication machinery
give some examples of extracelluar obsticles which can induce replication stress
UV damage
chemotherapies
other carcinogens like cig smoke
how does UV introduce replication stress?
causes chemical changes in the structure of bases which slows and stalls forks
how do you measure replication stress?
DNA fibre method
how does DNA the DNA fibre method work?
you incubate cells with CLDU for 20 mins then after exposure to IR incubate with IDU
then link flourescent antibodies to the 2 different analogues to visualise the DNA fibres
how will the DNA fibres look if the replication forks have not stalled?
:)
what does a stalled fork look like in the DNA fibre method?
fibre on the right
what happens if the topoisomerase enzymes arent properly resolved
causes lagging chromosomes or anaphase bridge development
what is an anaphase bridge?
when the sister chromatids dont correctly seperate during anaphase
what can come from anaphase bridge formation
micro-nuceli
describe the karyotype of most cancers
the karyotype is normally abnormal because of chromosome abnormalities and instabilities
- can cause chromosomal rearangements and genetic loss
what % of cancers have chromosomal instabiliy
60-80%
what can eventually happen with continous anaphase bridge and micronuclei formation?
mitotic catastrophy
what normally causes replication fork stalling?
DNA damage on the template which means that DNA polymerase is unable to continue
what happens to the DNA after a replication fork stalls?
it collapses into DSB
explain how replication fork stalling leads ot DSB
stalled forks cause maintained single strand break because the replication machinery was half way through replicaition (at which point there is a single strand break)
at the stall the replication machinery falls off leading exposed the SSB
endonucelases are attracted to the SSB causing the second strand to be cut
what happens if a replication fork runs into a break in the DNA such as a single strand break?
causes the fork to collapse in the same way as obsticles do
give the toxicity of small numbers and of large number of DSB's
small numbers are less toxic and more mutagenic
high numbers are very toxic to the cell and can activate apoptosis
compare the amount of replication stress in cancer cells to normal cells
replication stress is hightened in cancers compared to normal cells as oncogenes accelerate teh cell cycle and often reduce repair pathway activity
give a specific reason why cycling of the cell too fast can induce more replication stress
polymerase cant keep up because not enough nucelotides have been made
what type of replication stress will result from not enough nucleotides from being made?
will cause replication fork slowing and stalling + can induce DNA damage onto the template
why can replication stress happen often in stem cells
when they replicate faster than normal cells they can undergo nucelotide depletion which can cause replication fork slowing
what proteins recognise replication stress and triggers repair pathways?
ATM and ATR
which protein recognises DSB's
ATM
which protein recognises single strand breaks?
ATR
- RPA is recruited to the DNA to do this
what pathway is activated by the ATM protein after DSB?
the Chk2 pathway for either DNA repair or apoptosis
specifically how does ATM activate ChK2?
it phosphorylates Chk2 (because its a kinase)
what protein dictates if the cell will undergo apoptosis after DSB?
P53
what pathway is activated by ATR after SSB?
the Chk1 pathway
what form of DNA repair is performed by the Chk1 pathway
HR
explain the process of HR
RAD51 binsd to protein on the 3' overhang
RAD51 searches for the homologous sequence on the template
once found it recruits a D loop and forms a holiday junction
- the 3' end of the D loop is. used to continue replicating the stalled DNA
before RAD51 binds what needs to happen to the DNA during HR repair
the MRE11 complex needs to come and rescet the overhanging DNA
once the holiday junction and D loop is formed what enzyme is used to replicate the template?
DNA polymerase
what needs to happen after the template is replicated?
the Holiday junction needs to be resolved
what proteins resolve HJ's?
EME1 and other proteins
how can you visualise DNA damage and how can you visualise replication speeds?
immunohistochemistry of DNA damage proteins like gamma H2AX and RAD51
fibre analysis used for replication speeds