Lecture 14 - Maintenance of Genomic Integrity Flashcards
How what 3 ways can DNA damage occur?
- copying error during DNA replication
- spontaneous depurination
- exposure to different agents e.g. background ionising radiation, UV light, tobacco products
What are the 5 major types of DNA repair?
- direct reversal of damage
- base excision - corrects DNA damage caused by reactive oxygen species deamination, hydroxylation, spontaneous depurination
- Nucleotide excision repair - removes adducts that produce large distortions in DNA
- Homologous recombination repair and non homologous end joining - repairs DNA double strand breaks
- DNA mismatch repair - repairs copy error made during replication
Name types of DNA damage
- 7-methyl guanine
- O6 alkyl guanine
- UV induced thymine dimers
-
- enzymatic reversal
- removal and replacement of damage
What are the three DNA damage substrates and their repair enzymes that can reversed?
- UV dimers reversed by photolyase and visible light
- O6 alkyl guanine reverse by alkyl transferase
- DNA strand break reverse by ligases
What are the 3 substrates for base excision repair?
- alkylated cytosine
- spontaneous hydrolytic depurination of DNA
- formation of DNA adducts after exposure to reactive small metabolites
Describe the process of base excision repair
- the altered DNA base is excised in free form by a DNA glycosylase
- the resulting abasic site is corrected by the concerted actin of:
- an apurinic endonuclease
- a DNA polymerase
- a DNA ligase
What are the four steps of removal and replacement of a damaged base?
What enzymes are involved in each step?
- Removal = DNA glycosylase
- removal of apurinic site = apurinic endonuclease
- addition of new nucleotides = DNA polymerase
- ligation = DNA ligase
What dose nucleotide excision repair operate on?
What does it recognise?
What does it remove?
- dsDNA, cannot act on ssDNA
- it is non-specific, it recognises general distoriotns rather than specific adducts
- will remove and repair large adducts e.g. thymine dimers
- it is very efficient and error free
What are the four steps enzymes of nucleotide excision repair?
- endonuclease
- exonuclease - removes several or tens of nucleotides
- polymerase
- ligase
What is daughter strand gap repair?
- dimers remain after ‘repair’
- this is really a tolerance mechanism
- dimers are removed later from he double stranded DNA by excision repair
What is xerderma pigmentosum?
- autosomal recessive disorder
- patients show extreme sun sensitivity
- patients develop many skin tumours
- cultured skin fibroblasts show increases sensitivity to UV light
- cells can be shown to have defect in DNA nucleotide excision repair
What is the defect in XP?
- all deficient in NER, some have problem with daughter strand gap repair, many show unscheduled DNA synthesis
- excision deficient XP patients there is failure to excise the damage
- therefore the thymine dimer remains in situ
Describe the steps of nucleotide excision repair
- XPC (+XPE) recognise dimer
- XPB, XPD recruited to unwind DNA (helicases)
- XPF and XPG cleave
- polymerisation
- ligation
Mutation and Cancer in XP
- XP cells show a high mutation rate
- mutation probably due to unexcised dimers and, therefore, incorrect bases incorporated opposite damage
- this mutation represents a step towards cancer development
Which gene is often mutated in XP?
PTCH1 (patch one)
What are some of the variants of XP?
- not deficient in nucleotide excision repair
- not very sensitive to killing by UV, but cells are hypermutable
- sensitive to UV can be enhanced by caffeine
- defect in replication of DNA following UV exposure of cells (daughter strand gap repair)
- deficient in an enzyme DNA polymerase h, which is able to replicate DNA past UV photoproducts - translation synthesis
What main repair mechanism of BRCA1 and BRCA2 involved in?
double strand DNA break repair
What are the differences in primary sequences of BRCA1 and BRCA2?
BRCA1 = BRCT domains are found in many prairie proteins, as pairs of with an FHA domain BRCA2 = BRC repeats mediated binding to Rad51
What findings suggested that BRCA1 and BRCA2 were involved in DNA DSB repair?
- increased gamma ray sensitivity of BRCA1/2 -ve human cells and mice cells
- increased sensitivity to gamma ray sensitive suggests a defect in DNA DSB repair
By what two processes can DSB’s be repaired?
- non-homologous end joining (NHEJ)
- homologous recombination repair (HRR)
Describe homologous recombination repair and how BRCA2 and BCRA1 are involved
- Rad51 coats ssDNA to form nucleoprotein filament that invades and pairs with homologous DNA duplex - initiating strand exchange
- availability and activity of Rad51 is regulated by BRCA2
- BRCA2 binds to Rad51 through the eight BRC repeats in BRCA2
- BRCA2 controls intracellular movement and function of Rad51
- release of Rad51 is triggered by DNA damage by phosphorylation of Rad51 or BRCA2
- BRCA1 is also required for HRR
- mechanism through interaction with and removal of 53BP1 at sides to DSB, prior to resection and recombination
Describe the steps of non-homologous end joining
- recognising of DNA ends
- end synapsis
- end processing
- microhomology-based pairing
- flap removal
- gap filling and end ligation
Describe the characteristics of NHEJ
- Rad51 independent
- BRCA2 not required for DNA DSB
- V(D)J recombination is normal in BRCA deficient mice
- NHEJ is an error prone process
Give an overview of the roles of BCRA 1 and and BRCA2 in DNA damage responses
- BRCA2 occurs quite specifically through control of Rad51 recombinase in homologous recombination
- BRCA1 occurs in a much wider front, but links upstream sensing and signalling of damage, through 53BP1 in recombination repair
- BRCA1 also has roles in cell cycle checkpoints
How to PARP inhibitors work?
- used to treated HRR deficient tumour cells
- PARP1 usually acts to repair ssDNA breaks
- this is inhibited by PARP inhibitors
- when it reaches the replication fork, allows a dsDNA break forms
- this cannot be repaired in these tumours, cell death
What kind of an enzyme is Rad51
recombinase
What does DNA mismatch repair do?
repairs copy errors made during DNA replication
- mismatch repair genes reapir base-base mismatch
- and repaired insertion deletion loops which arise as a consequence of polymerase slippage during replication. slippage cases gains or losses in repetitive DNA. also called micro satellite instability
- genes that have microsattelites int heir coding region have an increase risk of mutation in HNPCC
What mutation is found in hereditary non polyposis colorectal cancer?
mutation in mistmatch repair genes
What is the mutator phenotype hypothesis in HNPCC?
- microsattelite instability - led to mutator phenotype hypothesis
- postulates that mismatch repair defects lead to mutation in other genes, including those known to play a role in the adenoma-carcinoma sequence
- therefore, the increased mutation rate is then the cause of accelerated tumurigenesis
- the mutator phenotype plays a role in tumour progression rather than in initiation
