Regulating DNA replication & DNA repair Flashcards
What sequence & base of that sequence are methylated in e. coli?
Why is there a delay from hemimethylated DNA in e. coli to fully methylated?
What are the steps from hemimethylated DNA to fully methylated DNA with the methylation of the daughter strand following DNA replication? 5 steps
GATC - A
Delaying the DNA replication process - full methylation allows new origin of replication to fire
- SeqA recognises & binds to heavily methylated GATC sites
- Dam methylase is blocked - inhibiting methylation of daughter GATC sites
- DnaA is blocked - in ADP bound state after DNA replication cannot bind
- SeqA slowly dissociates - in this time DnaA associates with ATP & Dam methylase can methylate GATC
- DnaA-ATP can then associate with methylated replicators at 9mers to allow DNA replication initiation
Why is DNA replication different in eukaryotes? 2 ways
What are the following components of eukaryotic DNA replication?
Cdc6
Cdt1
ORC
What are the steps occurring in the G1 phase for regulation of DNA replication?
What is different in the G1 phase to S phase?
What are the 4 steps in the S phase?
- Has a cell cycle - so can only fire once per replication cycle, whereas e.coli can fire more ori before previous DNA replication finished.
- can fire many origins at same time - e. coli only have 1
Cdc6 & Cdt1 = make up Mcm helicase loader
ORC = origin recognition complex
- Cdc6 binds to ORC
- Cdt1 loads Mcm helicase onto double-stranded DNA (pre-replicative complex of 2 helicases)
G1 phase has lower kinase activity - no activation of proteins
- Mcm is phosphorylated resulting in loading of Cdc45 & GINS complex onto Mcm helicase to complete 2x CMG helicase (complex upstream of OCR)
- ORC is phosphorylated, resulting in its inactivation (regulation - prevents more firing)
- resets on cell division
- Replication fork is ready - both CMG helicases load onto each single-strand of DNA & moves from origin displacing ORC
- DNA polymerase binds to both strands for replication
What occurs at the replication fork in prokaryotes?
How is it different in eukaryotes?
Which proteins are involved in leading & lagging strand synthesis in eukaryotes?
What protein do both prokaryotes & eukaryotes have?
DNA polymerase III holoenzyme moves as one flexible unit along the DNA but different subunits act connected together
Eukaryotic replication proteins function independently (not connected together)
Leading = DNA polymerase ε
Lagging = Started with DNA polymerase a, extended by DNA polymerase δ
DNA primase
In 5 steps, how does E. coli mismatch repair fix a mis-incorporated base/base pairing (hint: causes a kink in DNA helix)?
What is required of this process?
If the nick is made 5’ to the kink, what enzymes degrade misplaced strand?
If the nick is made 3’, what degrades it?
What is the eukaryotic mis match repair equivalent?
- Mut S functions as dimer - scans along DNA looking for kink in helical structure (not directly base pair mismatch)
- Mut S binds to kink & recruits MutL
- MutL activates MutH which nicks DNA near the mismatch
- Helicase UvrD unwinds DNA around nick
- Exonuclease digests misplaced strand & gap repaired by DNA polymerase III & sealed with DNA ligase
ATP for MutS, MutL & MutH to bind
Exonuclease VII/RecJ
Exonuclease I
Have homologues for MutL & S in own system
How do prokaryotes with Dam methylase detect old & new strands in DNA?
How do prokaryotes without Dam methylase detect old & new strands in DNA?
What are the 3 functions of methylation in prokaryotes?
What is the function of methylation in eukaryotes?
Are there other types of this?
Hemi-methylated DNA - daughter strand is not methylated
Use lagging strand as sequence to represent daughter - presence of Okazaki fragments similar to MutH nicking DNA strand for DNA polymerase III to repair & seal with ligase
- Regulate DNA replication
- Repair system using DNA replication machinery
- Restriction modification - defence mechanism against foreign DNA
Epigenetics - 5-methyl cytosine
Acetylation (histone) & non coding RNA
What sites are methylated in eukaryotic DNA?
What are they & how are they distributed?
What happens on the deamination of cytosine?
Deamination of 5-methyl cytosine?
What eukaryotic organisms do not have methylation?
CpG islands
CG sequences - non-randomly distributed/underrepresented unlike GATC
C -> uracil (larger error as not involved in DNA)
5C -> thymine
Invertebrates - drosophila
What are CpG islands?
What happens to the genes with a hypermethylated promotor?
Hypomethylated promoter?
What is gene-body methylation? (GBM)
What are the 2 results of this?
High density regions of CpG
Silencing of genes
Activation of genes
When the entire body of the gene from promotor to introns/exons has been methylated
- Increased level of expression/activation of genes
- Regulates alternate splicing due to methylation at intron:exon junctions
How does DNA methylation in eukaryotes work? 3 outcomes
What are the 2 ways to methylate DNA in eukaryotes?
When do these both occur?
What are the 4 steps of maintenance methylation?
What is de novo’s importance?
- Unmethylated DNA/chromatin results in more open conformation which is accessible for DNA-binding proteins & transcription factors
- Methylated DNA/chromatin results in impeded binding of transcription factors
- Methyl-CpG-binding proteins bind to DNA through methylated CpG binding sites (MBD) on stretch of CpG methylated DNA - act directly or recruit effector proteins
Maintenance methylation
2. De novo methylation
Maintenance methylation occurs every DNA replication/cell cycle. De novo happens after fertilisation due to erased methylation patterns
- DNA fully methylated before DNA replication at CpG sites
- In DNA replication, daughter strands not synthesised with methyl groups on CpG sites
- DNA is hemi-methylated
- Hemi-methylated parent DNA strand is recognised by DNA methyltransferase 1 (DNMT1) - binds to methylated CpG sites & methylates the CpG sites on daughter strand
Lays new methylation patterns after fertilisation for development
What is imprinting?
What is molecular basis imprinting?
IgF2 & H19 are clustered on the same chromosome. IgF2 stimulates growth, and H19 calms growth. If the male imprinting control region (ICR) is heavily methylated, what is expressed?
If the mother doesn’t have a heavily methylated ICR, what binds instead & what happens as a result?
What is the evolutionary advantage of this?
Form of epigenetic gene regulation that results in expression from a single allele in a parent-of-origin-dependent manner
Imprinted genes in a cluster together so share regulatory elements
Heavily methylated ICR means enhancers have access to IgF2 gene & stimulate growth
CTCF proteins bind - inhibiting access of enhancers to IgF2 so instead express H19 (calms growth)
Deleterious for mother to express IgF2 as will take all her nutrients & overgrowing of embryo
Which chromosomes are methylated in Backwith-Weidemann Syndrome BWS?
What is the result?
What about Silver Russell Syndrome/Thumbelina Syndrome?
Both - so bioallelic expression of IgF2 in both mother & father chromosomes
Large babies with overgrowth & abnormalities
No methylation of either chromosome means CTCF proteins bind - resulting in bioallelic expression of H19
What are the 2 types of spontaneous hydrolysis?
What happens in each?
What happens on deamination of the following bases & what do they pair to?
Cytosine?
Adenine?
Guanine?
Deamination & depurination
Deamination = loss of amino group through hydrolysis resulting in different pairs
Depurination = hydrolysis of beta N-glycosidic bond between N9 & ribose sugar resulting in deoxyribose without a base
Uracil - pairs with adenine (problem in DNA)
Hypoxanthine (pairs with cytosine)
Xantine - pairs with cytosine which is correct but bind with 2H instead of 3H
How does oxidation induce DNA repair?
What base is susceptible? What does it produce?
What binds to this product?
What kind of mutation does this cause?
What can be caused as a result of these mutations?
Presence of reactive oxygen species (ROS) with free radicals can oxidise bases readily
Guanine -> 8-oxo-guanine
Adenine
Transverse
Human cancers
Which DNA repair repairs deamination, depurination & oxidation?
On what basis does this DNA repair work on?
What are the 4 steps to this?
What is the downside to this?
How does Fail-Safe glycosylase repair DNA that has 8-oxo-guanine paired with adenine in its DNA? 2 steps
What else can glycosylases repair?
Base excision repair
Base has been incorrectly incorporated
- If a base has been incorrectly incorporated into the sequence, base excision acts on specific glycosylases enzymes - breaks glycosidic bond to remove wrong base
- Base is removed by enzyme specific for lesion
- AP site (abasic deoxyribose) is acted on by AP nucleases (endo/exo) to generate 3’OH for DNA polymerase I
- Abasic-ribonucleoside is removed & DNA polymerase I reincorporates correct nucleotide reading from the other strand
Not 100% accurate
- Glycosylase recognises incorrect pairing of 8oxoguanine:adenine & removes A
- Gives the cell a chance to repair 8oxoguanine
T:G mismatches due to deamination of 5-methyl-cytosine -> thymine
How does alkylation induce DNA damage?
What base is susceptible?
What does alkylation of this base do?
What does the product pair to?
How does direct repair repair alkylation in 2 steps?
Why is direct repair costly for the cell?
Inappropriate addition of an alkyl (methyl/ethyl) group to a base
Guanine with O6
Produces O6-methylguanine which produces a transverse mutation
Thymine resulting in G:C pair to A:T pairing
- Methyltransferase recognises the methyl group of O6-methylguanine
- Binds & removes the methyl & accepts it onto its own cysteine residue
Only occurs once per methyltransferase as expires after accepting the methyl group
How does UV absorption by nucleotide bases cause DNA damage?
How does this form?
Why is this particularly dangerous?
What kind of direct repair fixes this?
What are the 2 steps for this?
Results in formation of thymidine dimers causing kink in the DNA
Cyclobutane ring forms between C5 and C6 of neighbouring thymines in the DNA strand
DNA replication & polymerase machinery cannot pass - process stops & cell death
Photoreactivation
- DNA photolyase uses energy from light (light dependent to be ACTIVATED - uses energy) to break the covalent bonds to remove cyclobutane rings
- Correct sequence is restored & the enzyme dissociatest
