Genomic Instability and DNA repair Flashcards
Genomic instability
accumulation of unintended alterations to genomic sequence; elevated mutation rate
- muttaions don’t necisarily indicate genomic instability bc they can accumulate overtime via normal spontaneous mutation rate
Major forms genomic instability
- Gross chromosomal abnormalities (ploidy alterations, gene amplification, chromosomal structural alterations)
- Subtile sequence change ie nucleotide instability
(point muttaions, microsatelite instability)
Ploidy alterations
changes in number of chromosomes per cell
- aneuploidy- gain or loss individual chromosomes
- polyploidy- increase number of complete haploid sets of chromosome pre cell
gene amplification
increased copy number of gene or chromosomal region
chromosomal structural alteratiosn
inversions, deletions, translocations ect
Point mutations
- can change AA sequence of encoded protein -> modified activity
- can affect level gene expression, alter mRNA stability, change splice sites, impact initiation DNA replication
microsatalite instability
- changes in number repeats within repetitive sequence (run of repeated nucleotide can be prone to replication slippage)
genetic defects arise
b/c errors in fundamental processes or consequence of DNA damage 3 primary causes 1. Spontaneous events 2. Intrinsic Stresses 3. Extrinsic Stresses
spontaneous events
- physical breakdown (base lose, base deamination)
- Replication errors (base mis-insertions, replication slippage; polymerases not 100% active)
- chormosome segregation errors (mitotic process not 100% accurate)
- Telomere shortening
intrinsic stresses
oxidative stress from metabolism; leads to hydroxylation which affects base pair and leads to challenges for DNA replication
extrinsic stresses
- UV light (pyrimidine dimers, 6-4 photoproducts) and other environmental genotoxins
- cigaret smoke (bulky DNA adducts)
- Aflatoxin (DNA adducts)
- Chemotherapeutics (DNA breaks from adriamycin, strand crosslinks from cisplatin)
somatic mutation hypothesis
cancer arises b/c accumulation of mutations in growth regulatory genes but spontaneous mutation rate not high enough to drive cancer so Loeb theorized that genomic instability (increased mutation frequency) necessary for tumorigenesis which is consitant with most forms of cancer though it is argued by some that genomic instability is effect of tumorigenic growth not cause
- either way genomic instability plays causative role in at least some cancer by promoting mutation accumulation
Cell preservation of Genomic stability
Repair:
- Base-excision repair (BER)
- Nucleotide excision repair (NER)
- Recombinational repair (HR, EJ)
- Mismatch repair
- Cells cycle checkpoints
If can’t repair during transient cell cycle arrest inhinbiton of transcription, replication, chormosmal segregation -> apoptosis?
Double stranded DNA break repair
- produced by free radicals, various chemicals, replication across single-stranded DNA breaks
- most dangerous bc can lead to gain or loss of a lot genomic info
Mechanisms or repair:
1. non-homologous end joining
2. Homologous recombination
non-homologue end joining
-primarily in G1 stage cell cycle before exact copy of damaged site on sister chromatin available for repair
quick and dirty way to repair DNA break; recognize broken ends and ligate them back together; this is error prone
- critical for immunoglobulin gene rearrangement (intention breakage to genome to be repaired to get DNA rearrangement)
dx and non-homologue end joining
- not too much cancer, leukemia’s owing to Ligase IV mutation IDed
- Immunological defects
Homologous recomninatino
- used S and G2 which sister chromatids present
- break on one DNA molecule homologous intact template used to drive repair; ends invade intact template and get repair based on that; this is v precise
- highly conserved in organisms with 2x stranded DNA
- Core factors essential for viability
defects in homologous recomobination
associated with cancer predisposition, immunodeficiency and other developmental defects and chormosmal instability
ex. Brca1 and Brca2 regulate aspects of HR and bloom syndrome = bc mutation in helicase controlling frequency of recombination
Nucleotide excision repair fix
Lesions: bulky DNA lesions that distort helix, interfere with base pairing, typically obstruct transcription and replication
Relevance of nucleotide excision repair
mutations impairing NER associated with dxs of extreem sun sensitivity
- Xeroderma pigmentosum (associated with 1000 fold increase sun induced cancer)
- Cockayne syndrome
- Trichothiodystrophy
How does nucleotide excision repair work
Involved damage on 1 stand of DNA molecule and take advantage of 2 stands; recognize damage on one strand, remove damaged lesion so single stranded gap of DNA use intact strand as template for DNA synthesis and ligate back together
Base excision repair lesions
Small chemical alterations of bases these usually miscode but don’t typically block transcription or replication
Mechanism of base excision repair
- Damaged base flipped out of helix and DNA glycosylase claves it from sugar phosphate backbone
- Endonuclease recognizes basic site and cleaves sugar phosphate backbone (-> 1 of 2 types repair)
3.Short patch repair
OR - Long patch repair
Disease and base excision repair
mutations in base excision repair pathway not implicated in dx probably bc a lot of redundancy (many DNA glycosylases so defect in 1 gene not big deal)
mismatch repair pathway lesions
Mispaired bases, insertion, and deletion loops result of replication errors
mechanism of mismatch repair
- Lesion identified
- Additional MMR factors recruited to damage site
- Strand discrimination (determine which strand has correct sequence)
- Defective strand excised
- DNA re-syntehsized using intact strand as template
mismatch repair pathway and dx
- 5% colorectal cancers= hereditary nonpolyposis colorectal cancers = bc autosomal dominant dx bc mutations in MMR
- MMR pathway participates in meiotic recombination so defects -> infertility
Cell cycle checkpoints
aka DNA damage checkpoints
- ensure cell cycle events occur in correct order
- when genome damage occurs coridinte DNA repair activities with cell cycle progression (arrest cell cycle progression and promote DNA repair)
- checkpoints also impact effectiveness of cancer therapies which often act by causing DNA damage
how does DNA damage checkpoint pathway function
As singnal transduction cascade; detects presence of genome damage and relays that info to targets involved in DNA metabolism (core cell cycle machinery, DNA replication apparatus, DNA repair proteins)
Classical mammalian DNA damage checkpoint pathway vs second independent mammalian checkpoint pathway
Classical responds to 2x stranded DNA breaks
second- repsonds to replicative inhibitors that produce bulky DNA lesions
Classical mammalian DNA damage checkpoint pathway
Responds to 2x stranded DNA breaks; headed by protein kinase ATM which phosphorylates down stream checkpoint proteins like p53 which transduce DNA damage signal and up regulates transcription targets (p21,Bax) -> cell cycle arrest or apoptosis
Cell cycle checkpoint defects
lead to accumulation genome damage leading to defects and tumorigenesis
- mutations in Atm -> ataxia telangiectasia which has striking cancer predispositions and developmental defects
- Mutations in p53 in most human cancers
DNA damage checkpoints respond to DNA damage
that arises during cancer initiation and progression
activated oncogenes trigger DNA damage when
- they stimulate aberrant cell cycle progression; DNA replication defects and excess ROS production contribute to oncogene-induced DNA damage
- cancer cells appear highly dependent on stress response mechanisms that allow them to cope with high levels of stress therefore use drugs to target stress response pathways to target cancer
Philadelphia chormosome
translocation between long arms chormosomes 9 and 22 (puts BCR gene immediately next to ab gene and get a fusion bcr/abl which makes abl tyrosine kinase hyperactive driving proliferation and blocking cell death; Bcr-Abl = target of Gleevec which = kinase inhibitor); this translocation seen in almost all patients with chronic myelogenous leukemia
radial chormosomes
abnormal associations between non homologous chromosomes (try to do DNA repair with wrong DNA template)
Substitutions (type of subtile sequence alteration)
transitions- purine -> purine
transversions- purine -> pyrimidine
DNA decay
- sites of hydrolytic deprivation result in abasic sites which block replication and are cytotoxic
- other sites for hydroysis
- sites of oxidative damage
Base deaminiation
Affects several bases
Cytosine -> uracil (not a problem until it tries to replicate and have nucleotide change after DNA replication)
Adenine -> hypo-xanthine
Guanine -> Xanthine
5-Methycytosine -> thymine (challenge for cell to know if T should be there or if its there bc of spontaneous demanination making it hard for DNA repair machine)
frequency of spontaneous events
DNA is stable but these happen daily because genome is so massive
UV light
form of extrinsic stress:
- energy from UV light bosomed by DNA and affects adjacent pyrmiadine residues
- Pyrimidine dimers- adjacent pyrmindines become linked and form 4 membered ring shape
- 6-4 photoproducts adjacent pyridines 6 on one covalently links to 4 on adjacent base leading to diff structure which is highly distorting to DNA and messes up DNA replication
Biological consequences of genomic instability
- impaired cell survival; developmental defects
- Aging
- Increased tumorigenesis
- Inaccurate genetic transmission
Genomic instability disorders
inherited disorders of processes that usually repair damage have 3 classical features
- developmental defects
- premature aging
- cancer
Does genomic instability drive tumorogenic growth
genomic instability doesn’t directly drive tumorogenic growth oncogenes and tumor suppressors do
- Genomic instability isn’t gate keeper for proliferation it is care taker for gate keeps so can lead to issues with gate keeper but isn’t the gatekeeper itself
Most mutations are actually
harmful
specific conditions need to be met to get cells to clone and lead to cancer because have to have enough instability to drive cancer but not so much that it leads to cell death
Cell response to DNA damage
- Reversal of DNA damage
(ligation DNA strand breaks) - Excision of DNA damage (remove damaged strand and resent based on intact strand)
- Tolerance DNA damage
(damage arises during DNA replication use alternative DNA replication pathways and go back and fix this later)
what happens if spindle assembly checkpoint fails
- go into anaphase not properly attacked leads to anylpoidy when cells separate (mutations in spindle checkpoint components found in small number human tumors)
DNA damage checkpoints
Signal (abberant DNA structures) -> sensors -> transducers -> effectors -> cell cycle arrest, DNA repair, replication fork stabilization)
Ataxia Telangiectasia phenotype
Caused by deffect in Atm
- Cerebellar ataxia (neuronal degeneration)
- telangiectasisas (veins appearing on eyes and skin)
- cancer predisposition
- hypogonadism infertility
- premature aging
Ataxia telangiectasia cellular phenotype
Deffect in Atm: - chromosomal instability - radiation sensitivity - cell cycle checkpoint defective (Atm phosphorylates p53 which actiaves p21 and Bax)
atm pathway signal sensor transducer effectors when fx
Signal: DNA break
Sensor: Atm
transducer: p53
Effectors: p21 and Bax
if Atm not fx this pathway doesn’t fx properly
checkpoint deficit cell ionized radiation exposure
Cell lacking p53 exposed to ionized radiation ignores DNA damage and doesn’t induce cell cycle arrest can
-> mitotic fatal error and cell death; in rare instances lower levels of damage or more apoptotic machines defective can -> cell survival and cell dividing and developing more mutations and tumorogeneiss
genomic instability as driving source
- genomic instability leads to muttaions
- checkpoints try to deal with mutations so don’t get mutations
- once you have mutations oncogene induced damage triggers checkpoints -> apoptosis or cell cycle arrest as protective barrier ,but if mutate genes that respond to oncogene damage can lead to progression and malignancy
telomere related instability
- cells have hard time replicating ends of chromosomes which they get around using telomerase but somatic cells don’t have telomerase so each cell cycle the chromosome gets shorter until telomere that is normally hidden inside cell in T loop structure can’t be in T loop anymore so telomere stick out and trigger DNA damage response -> senescence this = protective mechanism which prevents unlimited replication
Unlimited replication and telomeres
get around telomere shortening by mutating p53 and Rb
Breakage fusion bridge cycles
telomere shortened and DNA end of two chromatids exposed so ends fuse together making one long DNA molecule with two centromeres when this goes into mitosis centromeres attach to different poles and this chromosome will be pulled apart and you get two different ends and then these can fuse and this is break-fusion bridge cycles; this leads to high levels DNA damage; cancer cells will eventually turn telomerase back on to allow continued proliferation because can’t tolerate this high level of DNA damage forever
