DNA repair and recombination Flashcards
What can potentially damage DNA integrity
Chemicals - water, uv light, reactive oxygen species etc
Modifications (alkylation)Replication errors
Etc
What types of spontaneous DNA damage usually occur
Hydrolytic attack
Oxidative damage
Non enzymatic methylation
Methylation of single stranded DNA (ssDNA)
What types of chemical can occur to individual bases
Base oxidation (e.g. guanine oxidation)
Depurination (sugar or nucleotide)
Deamination (NH2 removed)
Name some DNA repair pathways and any major implications in cancer
Base Excision repair (BER)(colorectal cancer)
MisMatch Repair (MMR)(HNPCC)
Nucleotide Excision repair (NER)(skin cancer)
Single Strand Break Repair (SSBR)
Lesion Bypass
DNA Double Strand break repair
DNA interstrand cross link repair
How can we find gene mutations associated with DNA repair
Positional cloning of gene that corresponds to the human disease.
What is the mechanism of Base Excision Repair (BER)
DNA Glycosylases create abasic/aurinic/apyrimidinic (AP) sites (replaces a nucleotide).
Activity signifies damaged bases at the site of insertion.
AP site recognised by AP endonuclease 1.
Cleaves the DNA phosphodiester backbone.
Polymerases fill the gap. Can either fix the single nucleotide (short patch repair), or fix the whole section (long patch repair)
What happens when Base Excision Repair (BER) doesn’t work properly
Causes ‘mutator phenotype’
Spontaneous mutations appear far more often
Bad because ^ causes cancer (specifically head/neck/naso-pharyngeal cancer).
What is the mechanism of Single Strand Break Repair (SSBR)
DNA damage recognition.5’ end: APTX (Aprataxin) removes AMP.
Polynucleotide 5’ kinase adds a phosphate group
3’ end: Polynucleotide kinase 3’ phosphatase removes the phosphate group.
TDP1 (Tyrosyl-DNA-Phosphodiesterase) removes products associated with abortive topoisomerase reactions.
What is the difference between TTRAP and TDP1
They both possess 3’ tyrosyl DNA phosphodiesterase activity, but TTRAP also possesses 5’ activity
Name a disease associated with Single Strand Break Repair (SSBR) genes
Recessive spinocerebrellar ataxias (uncoordinated movement/gait)
What is the mechanism of Mismatch Repair (MMR)
MMR caused by errors during DNA replication or damage to one base in a pair.
Damage recognised by MutS.
Strand incision by MutL.
Repaired by exonuclease (EXO1)
Name a disease associated with Mismatch Repair (MMR)
Hereditary nonpolyposis colorectal cancer (HNPCC).
What is the mechanism of Nucleotide Excision Repair (NER)
Fixes UV induced damage (e.g. reactive oxygen species)
XPA senses DNA damage.
XPF/XPG nucleases remove damaged DNA.
Replicator factor C/polymerase target the DNA for repair.
Repair synthesis occurs.
Name some diseases associated with defects in UV repair
Xeroderma Pigmentosum (XP) - high risk for skin cancer; atrophic skin; accelerated neurological degeneration Cockayne Syndrome (CS) - growth failures; neurological problems; retinal degeneration (but immune to skin cancer...)
What is the mechanism of Translesion Synthesis
Specialised DNA polymerase (TLS polymerases) replaces the normal DNA polymerase. Allows for a larger active site recognition, so can accommodate damaged bases.
For TLS polymerases to work, PCNA needs to be ubiquitinated/degraded.
What is the mechanism of DNA Double Strand Break (DSB)
Needed when both nucleotides break; DNA is broken in two.
Either:
Non-Homologous End-Joining (NHEJ) or;
Homologous Recombination (HR)
What is the Non-homologous end-joining (NHEJ) mechanism for Double strand break (DSB) repair
Error prone.
Ku 70/80 bind to DNA ends.
Ligated together by Ligase IV
What is the Homologous Recombination (HR) mechanism for Double strand break (DSB) repair
Error free.
Requires intact template for repair
Used in meiotic recombination
How does the cell decide on the type of DSB repair used?
Depends on which stage of the cell cycle it is in.
Name some diseases associated with defects in NHEJ mechanism for DSB repair
Human Severe Combined Immune Deficiency
Human Immunodeficiency with microcephaly
Name some diseases associated with defects in HR mechanism for DSB repair
Ataxia Telangiectasia (ATM) Nijmegen Breakage (NBS1) Familial ovarian and breast cancer (BRCA1/2) Bloom (BLM)
Name two crucial steps in recombination
Homology search (looking for homologous sequences Strand invasion
How is DNA recombination in mitotic cell facilitated
By cohesins (rings that hold sister chromatids together)
What occurs in meiosis 1 (the first of two cell cycle divisions)
Homologous chromosomes pair up; recombination occurs; all chromosomes segregate.
What is the issue with homeologous sequences (nearly homologous)
Can lead to chromosome translocations if occurs on a different chromosome.
Can lead to deletions, insertions, duplications or inversions if it occurs with a homologous chromosome.
How does recombination occur in mitosis/meiosis
DNA Double Strand Break (DSB) occurs.
Fixed using Homologous Recombination (HR).
Both 5’ ends are chewed back.
Homologous strand invades (D-loop formation).
Invading (broken) strand extended using other strand as template.
The other end forms a Capture Holliday Junction.
What is a Holliday Junction
Forms during recombination. Is the point where the two different strands intersect.
Has 4-way symmetry.
A meiotic recombination intermediate.
How is a Holliday Junction resolved
Cleaved by GEN1 resolvase
Either up/down or left/right. Direction dictates if a crossover occurs or not.
What organism can be used for genetic analysis of meiotic crossovers/gene conversion. Why
Neurospora crassa.
A fluorescent protein can be used to analyse recombination. Normal form is as a colony; a single cell wide. So easy to detect individual cell changes.
What is strand invasion
RAD51 recombinase loads on single stranded DNA and invades a homologous DNA strand. Facilitated by BRCA2 and RAD52
Why is a Holliday Junction Resolution important
If unresolved (D-loop dissembled), then no crossover occurs.
What is DNA Cross Link Repair
Cross links cause genome instability.
Name a disease caused by DNA cross linking / genome instability
Fanconi anaemia:
High cancer risk (leukemia)
Stem cell failure in bond marrow
Growth retardation and abnormal development
Describe the fanconi anaemia repair pathway
FA complex recruited.
FAND2 ubiquitinylated
DNA repair factors recruited
Linked DNA unhooked (by MUS81/XPF1 nucleases)
Translesion synthesis occurs
Excision by the NER pathway. DSB end resection.
Homologous recombination occurs.
