Lecture 10 - DNA repair systems and SOS Flashcards
What are the five main repair systems?
Homolgous recombination (by dHJ pathway, SDSA, BIR) Non-homolgous end joinging Mismatch repair Nucleotide Excision Repair Base excision repair
What does UV light do to the genome?
Crosslinks adjacent pyrimadines forming
-cyclobutane pyrimadine dimer (some replication complexes can read through)
OR
-6-4 photoproduct
What is the process by which a photodimer may be reversed?
By direct repair
- UV light induces crosslinks
- light induces photoreactivation, CPD photolysase reverses the crosslink
- bases are separated
What are the two pathways that can begin nucleotide excision repair?
- global genomic repair (GGR)
- transcription coupled NER
When does transcription coupled NER act?
Operates specifically when transcriptional complexes are stuck at a site of damage
What happens in transcriptional-coupled NER?
1) RNA polymerase reaches a site of damage
2) proteins (CSA CSB) recognise stalled transcription complex and bind to it
3) CSA and CSA either removed stalled transcription complex or push it back
4) TFIIH cleaves RNA polymerase CSA and CSB
5) (two pathways converge) TFIIH recruits a multiprotein complex and helicases unwind DNA
6) Incision of DNA around the site of damage
7) synthesis and ligation of new DNA by sliding clamp protein and bypass polymerase (high fidelity Pol o-)
What bypass polymerase is used in nucleotide ecision repair?
high fidelity Pol o-
What occurs when have a mutation in XP (of the general excision repair pathway)?
Xeroderma pigmentosum
- DNA damage not repaired but cell death not triggered
- mutations accumulate
- early onset of caner, most skin cancer UV induced damage
What occurs when have mutation in CS (of the transcriptional NER pathway)
Cockeyne syndrome
- stalled transcription complex not removed
- programmed cell death
- premature again, developmental disorders
What is the SOS system?
survive and accept mutations
What triggers the SOS system?
RecA recombinase regulates SOS
How is SOS system triggered?
1) RecA expression is repressed by the expression of the lexA gene - downregulates expression of RecA and of other target genes
2) Some RecA proteins are still expressed, and are activated in UV damage where bind to ssDNA
3) activation of RecA triggers lexA cleavage removing repression and allowing more RecA proteins and target gene to be expressed
4) allows rapid return to non SOS state
Aside from triggering the SOS sytem, what else does RecA do?
- carries out strand exchange in HR
- triggers the degredation of the phage lambda repressor, excises phage lambda from chromosome, more chance of surviving it phage leaves the bacterium to infect another
How can lesions at stalled replication forks be bypassed in SOS system?
By translesion synthesis
What is the process of translesion synthesis?
1) PolII and PCNA complex arrive at the side of damage and replication fork stops
2) RecA binds to ssDNA and is activated
3) Pol V (bypass polymerase) is recruited by RecA to replace polIII as has capacity to read through damage
4) PolV continues DNA synthesis only for a short period until past lesion and falls off
5) PolIII binds to site and continues synthesis
What is the purpose of translesion synthesis?
bypass lesions and able to continue replication but at higher mutation rate
Where do errors not matter?
In creation of antibodies, however where the errors occur does matter
What is clonal selection?
1) precuror cells in bone marrow proliferate and diversify leading to different B resting cells
2) undergo testing for competent proteins and education
3) in peripheral lymphoid organs antigen binding to specific B cell activates that B cell
4) Activation leads to proliferation and differentiation of B cells into antibody secreting effector B cells
What are the regions of antibodies?>
constant region
variable region
hypervariable region
What are teh two different types of constant region in the light chain?
K or lambda
What are the different types of constant region in the heavy chain?
alpha beta e gamma or mew
What is the immunoglobulin tetramer (H2L2) linked to?
two copies of the signal tranducing heterodimer (IGa and b) fixes antibody to membrane
What does the mechanism of Ig gene rearrangement invole?
RSS and Rag (recombination activating gene)
What is the mechanism of Ig gene rearrangement?
RAG1/2 cleaves single strand at RSS on random V and J segments, exposing a 3’OH group
2) 3’OH goup attacks the other strand to form a hairpin end (intramolecular transesterification) intervening DNA is removed
3) NHEJ repair hairpins together
What happens when the intervening piece of DNA is not discarded from intervening VJ region?
can be integrated into random regions in the genome , identifing sequence left and right of the intervening section (repeated because formation of staggered breaks) with Rag1/2 acting as a transposase
How was the immune system originally evolved?
By a single transposition event inccluding RAG genes integrated into primordial germline receptor
What is the mechanism by which the different VJ segments join
1) Artemis cleaves hairpin ends imprecisely, producing protruding ends
2) protruding ends can be trimmed randomly leading loss of few bp, also can be filled in by polmerase (p nucleotides)
3) terminal deoxynucleotidyl transferase TdT inserts ‘N’ nucleotides (only experssed in lymphoid cells)
4) ligation leading to diversity
What is class switch recombination?
constant region of antibody shuffling to change fucntion
-AID indirectly creates dsbreaks at switch regions (AID targets) between the different constnat region cassettes
What does AID do?
Activation induced cytidine deanimase acts on exposed single strand (e.g where transcriptional complexes move through)in transcribed regions to deanimate cytosine to uralcil by addition of H20
How does AID indirectly induce DSB?
1) Uracil reisdues inserted into DNA sequence by AID
2) UNG from BER system removes theU base but leaves the back bone intact
3) APE cleaves DNA strand at sites where base is lost
4) APE and UNG together lead to the conversion of some dUs to abasic sites and some to SSBs
5) DSBs can occur when two breaks occur nearby on opposite strands
6) MMR proteins bind U:G mismatch and recruit error prone (pol eta) exonucleases resection to creat DSB
7) DNA polymerase fills in gap to create blunt/nearly blunt DSB
What DNA changes does AID deanimation predict?
high incidence of C/G to T/A transitions as tje repair system is not accurate and U often not repaired
What base changes occur in SHM?
ALL types of base changes
When does SHM occur and whay?
after VJD recombination and to increase antibody diversity
How was AID shown to be a mutagen?
expression of AID in e.coli led to a high number of colonies idicative of high mutation rates, some of which led to antibiotic resistance
also expressed AID without UNG glycoslyase led to even higher rate of mutations if BER inactive
What does expression of AID at wrong time in lymphocytes and in other cells types mean?
inc mutation raes and increased risk of cancer
What are otehr cytidine deanimases related to AID?
APOBED
What have signatures of mutational processes in human caner shown?
That APOBECs appear as one of the main casues of mutations leading to cancer
