DNA damage and repair
What are some sources of DNA damage?
ROS, UV light, chemicals, X-rays
What does hypermethylation in cpg islands mean?
associated with abnormal gene silencing (caretaker genes): recruitment of histone deactylases and histone methyltransferases, heterochromatin formation, inhibition of transcription, this is mechanism for inactivation of tumor suppressor genes in addition to mutation global hypomethylation sometimes results in loss of imprinting, causing imprinted genes to be overexpressed
How does methylation work in inherited cancers?
autosomal dominant: one good copy, one bad, inherited bad copy is 1st hit 2nd hit: something happens to good copy in somatic cell, 2nd hit might be DNA methylation can't be first hit in inherited cell because DNA methylation is not inherited, erased in germ line cells
What happens if there is defective DNA repair?
cancer
Describe microsatellite instability.
change in any length due to either insertions or deletions of repeating units in a microsatellite within a tumor compared to a normal tissue, global at least 2-5 loci unstable caused by defects in mismatch repair system, can be due to: inherited (lynch syndrome) or somatic mutation or methylation of genes encoding MMR pathways
What is cpg island methylator phenotype?
characterized by widespread promoter hypermethylation present in colorectal cancers
People with lynch syndrome have increased risk for which types of cancer?
colon, endometrial
What is base excision repair?
damage to a nitrogenous base portion of nucleotide glycosylase removes base, backbone intact AP endonuclease cuts backbone, 5' of AP site (created when base removed) and removes the deoxyribose sugar DNA pol adds new nucleotide DNA ligase seals
What is mismatch repair?
deals with mismatched nucleotides, structual error, non complementary, not base paired right, no actual damage (repairs polymerase error and small insertions or deletions), but needs to figure out which strand is wrong important for stability of microsatellites mutations of MMR enzymes associated with inherited colorectal cancer
What do DNA glycosylases in BER do?
family of enzymes that recognize specific types of damaged nitrogenous bases deaminated C, A alkylated or oxidized bases bases with opened rings bases with reduced double bonds catalyze removal of the damaged nitrogenous base by hydrolysis of N glycosidic bond
Describe how cancer and genomic instability occur.
faulty DNA repair mechanism leads to mutator phenotype, damage accumulates and creates mutations, if mutation of tumor suppressor genes, lose control of cell cycle check points, cell continues to proliferate even with damage, more mutation occur DNA repair proteins: care takers or tumor suppressors
What is a double stranded DNA break?
happen in every S phase, can also be caused by ionizing radiation, oxidizing agents lesion leads chromosomal trans-locations and cell death if not repaired promptly (worst type of damage), bad because naked DNA at break site vulnerable to nucleases DNA fragments joined could be broken ends of same chromosome or ends of 2 chromosomes (might be putting together wrong chromosomes)
What does HR require?
homologous sequences, strand invasion, strand elongation, cleavage and ligation mitotic: no crossing over usually
How is a collapsed replication fork repaired?
if replication fork reaches single stranded break, it collapses (effectively creating double stranded break) HR can be used to repair the form using newly synthesized DNA from the other strand
What happens if the rate of DNA damage does not equal the rate of DNA repair?
leads to pathology pathology/damage can lead cell to become senescent or undergo apoptosis (cancer can occur)
How does a defect in the MMR pathway cause microsatellite instability?
loop of mismatched DNA from slippage with MMR, recognizes hairpin loop and removes it to restore original sequence with defect in MMR: DNA replicated, may get expansion or deletion depending on which strand hairpin loop is on
What are the different pathways for DNA repair?
mismatch repair base excision repair nucleotide excision repair homologous recombination non homologous end joining
How does homologous recombination work in meiotic cells?
mitotic: one strand invades copy of the DNA, released and rejoins damaged copy meiotic: both strands get captured on copy of DNA and synthesized, there is exchange of DNA (crossing over)
What are the subtypes of excision repair?
most common type of repair in human cells subtypes: base excision repair, nucleotide excision repair: (transcription coupled repair, global genome repair)
Why does genetic anticipation occur?
mutation itself is changing through generations of the family
What does expansions of trinucleotide repeats cause?
neurological or neuromuscular disorders, dynamic mutations, change over time, characteristics of disease changes over time: increased odds of inheriting, decreased age of onset, increased severity in later generations, called genetic anticipation expansion of particular microsatellites in particular genes causes particular diseases no overall problem with microsatellites in general
How does homologous recombination work?
nucleases at site of break digest 5' end back, so 3' overhang, 3' overhang invades copy of homologous DNA, DNA synthesis based on other undamaged copy, then re anneals to other damaged strand, uses copy to re synthesize part that was damaged
Changes in methylation can occur in BLANK?
other diseases besides cancer
What is homologous recombination?
precise, no nucleotides lost dominant in S and G2 phase accurately repairs back to original sequence, but more complicated and needs a copy of the DNA homologous copy provided by sister chromatid (genetic exchange bw pair of homologous DNA sequences) used to restart stalled replication forks involved in crossing over during meiosis, but different because of resolution of joint molecule
What is xeroderma pigmentosum?
rare autosomal recessive disorder caused by defects in proteins involved in NER UV light causes pyrimidine dimers, NER is pathway that primarily fixes this so increased risk of CA, increased photo-sensitivity this disorder was studied to determine how NER pathway works, took samples of cells from families that had same disease phenotype, cultured cells and they were complementary groups (combine to restore normality/functioning), meaning that each family had mutation of a different enzyme in the pathway (locus heterogeneity)
What are the basic steps of excision repair?
recognize damage remove damage by excising part of the damaged strand fill in gap, using other strand as template ligate to seal nicks
What does global hypomethylation of heterochromatin regions with area of repeats?
recombination due to formation of euchromatin, repeated sequences tend to stick together and recombine, causes genomic instability and cancer
What does it mean for the genes affected by trinucleotide repeats to be polymorphic in nature?
several versions of gene not associated with disease people with disease have version of gene with more repeats length of repeat associated with severity of disease repeat sequences rich in cg, methylation, gene silencing
What is the chromosome breakage-fusion-bridge cycle?
shortened telomeres- triggers p53 checkpoint, leads to senescence no p53- NHEJ pathway sticks all the ends together, can't tell which ends belong together, sticks 2 chromosomes together, have 2 kinetochores, these are pulled apart, then eventually have new double stranded breaks, stuck together again and endless cycle if have telomerase reactivation - leads to cancer
How did trinucleotide repeats get expanded?
slippage of newly synthesized strand during replication: cg tendency to form hairpin loops, DNA pol copies same area twice, should be repaired by mismatch mechanisms, but assumed structural features of repetitive DNA make this more difficult, but nothing wrong with repair pathway, works for all other microsatellites Recombination: repetitive sequences more likely to undergo recombination, expansion can result due to unequal cross over and or due to pol slippage during the repair DNA synthesis (DNA synthesis done by different pol that might be more likely to slip back)
What are the subtypes of NER?
transcription coupled repair: removes lesions from the template strand in transcribed regions of DNA, triggered by stalled RNA pol, targets DNA repair to genes being actively used global genome repair: not targeted, depends on enzymes being around, recognizing damage removes lesions all over genome decreased in terminally differentiated cells, cell doesn't need to waste energy to repair DNA it's not using.
What does hypermethylation of cpg islands in the promotor regions of genes cause?
transcriptional repression, loss of expression of tumor suppressor genes, leading to cancer both hypomethylation and hypermethylation contribute to cancer
What genes are involved in formation of the joint molecule in HR and can lead to breast cancer?
BRCA1 and BRCA2
How do DNA methylation changes occur in cancer?
CpG islands found in many promoters normally not methylated, while most in other areas of genome usually are abnormal methylation patterns in cancer: overall hypomethylation or hypermethylation --> leads to abnormal gene silencing and decreased transcription
What types of genes affected by MSI?
DNA repair, signal transduction, apoptosis, TS regulation, immune surveillance mutations in a lot of genes that can affect functioning of cell and contribute to cancer
How do non homologous end joining work?
Ku dimer activated by kinase, Ku (helicase) and nuclease (artemis) binds ends so there are regions of homology (areas that can base pair), extra DNA trimmed and ligased back together can't restore DNA to how it was, but stops large deletions and hopefully it puts the correct DNA back together error prone, several base pairs of site of break removed
What is locus heterogeneity?
Mutations at different loci can cause the same phenotype
What does deamination of the following bases lead to?
adenine: hypoxanthine guanine: xanthine cytosine: uracil thymine: no deamination methyl c: thymine (harder for cell to detect and repair)
What are characteristics of cancers that have MSI?
affected cells usually diplod, rarely show loss of heterozygosity assicuated with modestly improved prognosis such tumors may respond differently to chem and radiation: 5 fluorouracil selectively kills cells with MMR system intact, no benefit for patients with MSI tumors, may respond better to immunotherapy
Why does DNA need to be repaired before replication?
can lead to mutation one strand is damaged, one normal, so when replicated, have cells will be normal and half will be mutated
What are the types of nucleotide structural damage?
covalent attachment of large hydrocarbons pyrimidine dimers damage involves at least 1 nucleotide, maybe more
What is nucleotide excision repair?
damage involves entire nucleotide, larger structural distortion helicase (TFIIH) unwinds damaged section of DNA double excision (cleavage on both sides of damage) DNA pol fills gap ligase seals nick
What are the types of DNA damage?
depurination: removal of purine nitrogenous base, spontaneous loss of adenine or guanine deamination: common, spontaneous loss of NH2, results in unusual bases not found in DNA, most often, helps with detection oxidative damage to nucleotides: ROS produced during metabolism
What is the difference between mismatch repair and excision repair?
different enzymes used in each pathway
Expansions in a non coding region?
fragile x, freidrich ataxia, spinocerebellar ataxias, myotonic dystrophy large changes in repeat number associated with maternal transmission Disease causing mechanisms: decreased TS due to change in chromatin structure or increased methylation (fragile X) RNA gain of function: abnormal interaction of RNA with proteins involved in mRNA processing (MD), mRNA proteins sequestered and not available to process other RNA
Expansions in an exon.
generally CAG repeat, which encodes glutamine huntingtons disease associated with paternal transmission disease causing mechanism: toxic gain of function of mutant protein (protein aggregates), glutamine likes to interact with other proteins, adheres with other proteins, interfere with functioning
How many accidental base changes results in permanent change (mutation)?
less than 1 in 1000 DNA double stranded nature makes it suited for repair
What does the mechanism of disease caused by trinucleotide repeats depend on?
location of repeats within the gene, and function of affected protein fragile X: repeat expansion in promotor/regulatory region, transcriptional silencing can be in intron, impaired transcription, loss of function huntingdon's: exon repeat, polyglutamine, gain of function, new properties to protein
How does mismatch repair work?
proteins scan DNA looking for nicks (nicks due to lagging strand), newly synthesized strand, has more nicks, more likely to be wrong area between mismatch and nick is excised DNA pol fills gap, ligase seals
What is non-homologous end joining?
quickly puts chromosome pieces back together, so no big deletions, less likely to have mismatched chromosomes dominant mechanism in G1
What are microsatellites?
repeating short sequences scattered throughout genome, more frequent in non coding regions most abundant class of repetitive DNA repetitive nature presents challenges to replication and repair mechanisms
