Unit 5 redone b/c last one is too long Flashcards
An example of direct repair would be?
Photolyase/photoreaction
Uv damage creates a cyclobutane pyrimidine dimer and this is removed
In most basic terms, what is excision repair?
when a damaged region is removed and synthesis of complementary strand replaces damage
Mismatch repair occurs after? And preferentially corrects what?
immediately following replication and it can recognize a mismatch in base pairs, deletion/ insertion loops
preferentially corrects newly synthesized strand/daughter strand (biased toward restoration of wild type)
how can mismatch repair recognize parent strand from daughter strand?
1) methylation state- daughter strand is usually not methylated and parent strand is methylated (hemimethylated state)
2) G-T mismatch from deamination of methylated cytocine preferentially removes T and replaces with C
What are the two forms of excision base repair? How do they differ?
1) Base Excision - removes a base (deamination/alkylation/uracyl glycosylase)
- short patch (removes 1 base)
- long patch (removes 2-10 bases around damaged site)
2) Nucleotide excision repair (NER) - removes bulky lesions (uv damage = photoreactivation in E.coli but NER in mammals)
- GG-NER
- TC-NER
What are the two types of damaged recognize by repair systems?
1) Single base change - this type of change affects the sequence only but does not change helix structure not does it affect transcription or replication. It will affect future generations
2) Errors in replication - A instead of C (AG) creates a distortion in the helix (example: methylated bases or depurination)
What kind of errors can distort the helix and affect replication and transcription? How are they repaired?
1) methyl group added to base - repaired by dealkylation
2) Depurination - removes purine -repaired by insertion
In recombination repair what is replaced and how?
replaces double stranded regions and gets the undamaged strand via recombination
also corrects DSB of homologous strands
What are the steps of excision repair in E.coli?
1) incision by an endonuclease (12bp on either side)
2) Excision by 5’-3’ exonuclease or a helicase will displace damage strand
3) Synthesis via a polymerase
4) ligase seals the nicks
What are the steps of the Uvr excision system in bacteria? What patch does this system prefer?
1) Uvr AB dimer recognizes bulky lesion
2) Uvr A dissociates from UvrB
3) UvrC joins UvrB to form a dimer
4) UvrCB dimer makes incision on either side of damage
5) Uvr D helicase displaces damaged strand
6) Polymerase I excises damaged strand
* Pol I can also synthesize or Pol II or III
* prefers short patch repair
What happens if there is stalled transcription in e.coli? how is Uvr implemented?
MFD will recruit Uvr when there is stalled transcription
-transcribed strands are preferentially repaired
What is XP? Xeroderma pigmentosum
Xeroderma pigmentosum, mutations in nucleotide excision repair (NER) genes. Patients w/XP cannot excise pyrimidine dimers and this causes skin disorder and susceptibility to cancer
In eukaryotes what are the two NER systems?
1 GG-NER
2 TC-NER
What recognizes damage in GG-NER?
XPC recognizes damage and initiates repair
GG-NER in eukaryotes recognizes many types of damages, which one does it struggle recognizing?
can’t recognize the pyrimidine dimer damage b/c lacks photolyase. A DDB will recruit XPC to the dimer
What recognizes damage in TC-NER in eukaryotes?
RNA Polymerase II recognizes damage. This repair system repairs lesions on transcribed parts of active genes.
In TC-NER and GG-NER what is the unwinding helicase?
TF2H is the unwinding helicase along with XPB and XPD
XPF and XPG do what?
are exonuclease in the TC/GG NER repair systems
In TC/GG NER what seals the nick
Ligase III and XRCCI
Which enzymes are responsible for base excision repair?
Glycolyases and lyases remove bases
What is the difference between glyolyases and lyases?
Glycolyases use DNA pol delta/epsilon to remove base and does this on long patch path
vs.
Lyases use DNA pol beta and remove bases from short stretch pathways
How does glycolyases remove bases?
will cleave bond between base and sugar sometimes it can use amino acids to open up sugar ring
Uracil and alkylated bases are recognized by what enzyme? And how are these repaired?
Through glycolyases
glycolyases uses a base flipping mechanism in which the base are flipped out of double helix, excised or modified, and returned to helx
What is error prone synthesis in e.coli
When DNA polymerase inserts nucleotides at random when it can’t find the complementary base
- DNA pol V (umuCD) - repairs UV damage
- DNA pol IV
What is a mut gene?
a gene that codes for a mismatch repair system that deals w/mismatch base pairs
mut genes also have a mutator phenotype in that they increase the spontaneous mutations
Mismatch repair system is biased toward restoration of what?
wil-type
In eukaryotes what is the MutS/L repair system
muts/l fixes errors caused by replication slippage
slippage creates a single stranded loop and mut S/L bind to mismatch
exonuclease, helicase, DNA pol, and ligase are involved in mismatch repair in muts/l
Recombination repair systems in e.coli are coded by what genes?
coded by rec genes.
after replication, there are gaps that are produced due to the opposite sequence having to be repair
Explain recombination repair
1) damage on one strand of DNA
2) when replication occurs the two daughter duplexes will be different
- one daughter will have intact parent and intact complementary
- the other daughter. will have a gap on parent
3) single strand from intact duplex is used to replace gap but this leaves a gap in duplex
4) gap is repaired by repair synthesis
In recombination repair system, what happens when replication fork stalls? what can resolve this issue?
Resolvase - targets holliday junction created between two duplex DNA
helicase will also help roll the fork and restore replication
How can resolvase create a DSB?
if it cuts either side of complementary strand. if damage is a nick it can also lead to DSB
What causes DSB in eukaryotes? What repairs it?
radiation and free radicals. Recombination is preferred in homologous but if stands are non homologous then NHEJ repair comes into play
what are the repair genes involved in repairing DSB?
Rad - named this way b/c of their sensitivity to radiation
Rad52 is required for recombination repair of DSB
What is non-homologous end joining NHEJ repair
in absence of homologous sequence, NHEJ repairs DSB by ligating blunt ends of duplex DNA
What are the steps involved in NHEJ repair?
1) recognition of broken ends by Ku 70 + 80
2) Ku binds to ends of DNA and forms a bridge between two ends of DNA
3) DNA strands are ligated by ligase V and XRCC
Failure to repair DSB can lead to what?
chromosome instability and increase chance of cancer
Why must chromatin and histones be considered in DNA repair of eukaryotes?
b/c chromatin impedes replication and transcription b/c nucleosome must be diplaced and DNA unwound
thus, histone modification and chromatin remodeling are essential for repair of DNA damage in chromatin
How are histones modified and chromatin remodeled?
by phosphorylation of H2A at DSB, phosphorylation stabilizes assembly of repair factors and modifying activities
Explain the steps of chromatin remodeling
1) damage occurs at chromatin
2) REMODELING - allows access to lesion
3) REPAIR - lesion is repaired
4) RESTORE - chromatin assembly restores original structure (remodelers and chaperones are needed to restore chromatin structure)
What is SOS and what triggers this system?
SOS is the global repair response in bacteria and it is triggered by RecA
RecA cleaves what?
LexA which is a repressor of operons and SOS
Inactivated LexA causes what?
operons and SOS to be expressed
Explain how RecA interacts with LexA and affects SOS
Inducing Signal > causes RecA to increase and this cleaves LexA, LexA no longer suppresses operons and SOS is activated
No inducing signal = equals no RecA = no LexA cleavage = repressor for operons = SOS shuts off
