G2 Flashcards
What is the luria and delbruck fluctuation testq
showed that survival of resitant colonies of of E.coli in presence of T1 phage was not because phage was inducing mutations. mutations occured at different times in different plates
phage was environmental selecting agent
what is replica plating?
lederberg - exact replica due to stamp on arg+ and arg- identifies arg mutants.
What doe mutation hotspots on lacl show?
- that there is a 5’methylcystosine at each hotspot which can be deaminated to thymine
- that CTGG is a major indel hot spot, repeated tandemly - addition or loss leads to mutation
what is depurination and rate in mammals
loss of pruine base
mammalian cells can lose 1-^4 per 20 hour gen.
what is replication slippage
section newly synthesized loops back on itself in ssloop DNAP copies region second time due to loop and in the enxt generation therefore get one with normal one with more repeats.
usually happens at repeats `
What are most liable to cross over unequallyy
strs
cross at ares of homology
how are photodimers created by UV fixed?
Repaired by CPD photolyase which binds to and split dimers = photoreactivation
(cyclobutane photodimer = CPD)
how is O-6 guinanine alkylation repaired?
alkytransferases remove groups
eg. methyltransferase AlkB family of deoxygenases.
how do DNA glycosylases work?
hydroylse sugar bonds releasing the altered base leaveing an abasis site
how does AP endonuclease work
cleaves damaged strand at 5drp of abasic site
(deoyribosephosphate)
how does AP beta lyase work
cleaves DNA at 3’drp of abasic site
how does AP beta delta lyase worjk
cleaves at 5’ adn 3’ phosphates around abasic site
how are holes left by removed abasic sites fixed
DNAP replaces excised
XRCC1 ligase alpha seals nick
Describe long patch BER
initiated by AP endonuclease - eg. APEX1 which cleaves DNA 5’ to abasic site, leaving 3’ hydroxyl group as substrate for DNAP beta
which fills removed base and several bases downstream displacing the strand on the 3’ side of cut.
displaced strand is removed by flap endonuclease and ligated by DNA ligase.
what does transcription coupled nt excision repair do?
repairs transcribed DNA regions
what does global genomic repair GGR do
correct lesions in any section of genome activated by stalled replication forks
describe tc NER
when transcription by RNAP stalled by lesions then CSA and CSB bind to complex
attracting TF11H (10 subunits) which contain helicases XPB and XPD
miscoding region cut out and replaced
describe GCR
damage sensor XPC scans for distorted DNA, recognises it and binds to opposite strand
XPC attracts TF11H via XPB ATPase activity.
- what does TF11H do?
opens DNA allowing recuritment of XPA and CDK activating kinases (CAK)
- what does XPA do?
allow release of TF11H and CAK and recruitment of RPA (XPD heicase activity is relased open the DNA further. XPC-CEN2 is released from the complex)
- what does RPA do?
coats the undamaged DNA strands
- what does the release of CAK faciliate?
recruitment of XPF-excision repair complementeing (ERCC1) complex and XPG
- what does XPF excision repair do?
carry out incision 5’ and 3’ in the damged DNA and DNAP fills gap. and XRCC1 ligates
Describe the process of DNA mismatch repair
- reconigtion of damage in enwly replicated DNA by MutS
- Binding of MutS to distrotions in DNA cuased by mismatches
- it recognises parent strand and nicks daughter strand - attracts MutL, MutH and UVrD
- MutH cuts the section with the incorrect bases and UVrD DNA helicase activity unwinds DNA.
- Error containing strand is degraded by ssDNA specific exonucleases (e.g Exo1, ExoV11) and resynthesized by DNAP
what non homolgous end joining NHEJ
major pathway for repairing ds breaks DSBR
Describe the process of NHEJ
- initiated by binding of Ku70 and Ku80 to DSBR
- this recruits DNAPKcs and aligns DNA ends
- DNAPK recruits endonuclease Artemis which trims the 5’ and 3’ overhangs
- PNKP is recruited via XRCC4 - LIG4 releases exisiting 5’p and 3’p residues
- XLF interacts with XRCC4 - LIG4 and DNAP
Describe homologous recombination
recognition of double stranded break by MRN complex
MRN associated CtBP which produces 3 ssDNA overhangs and 3ss DNA is stabilised by RPA
RAD52 promotes replacement of RPA with RAD51 to allow fromation of RD51-DNA nuceloprotein filaments
BRAC2 assists the assembly of RAD51 onto RPA ssDNA, RAD51 coated ssDNA enables strand invasion.
what is the first step in all BER reactions
removal of abasic site by dna glysoylase
what does TFIIH contain
10 subunits including XPB and XPD which are helicases
what organisms use BER
pro and euk
what are two diseases caused by defects in excision repair systems and how do they differ
xeroderma pigemetosum - has XPG defects
Cockayne syndrome = CSA and CSB defects
what does cockayne syndrome cause
dwarfism, deafness and retardation plus premature aging
