Unit 3 Flashcards
(30 cards)
Sources of DNA Damage
Replication errors
- Replicative polymerases have a 3’-5’ proofreading exonuclease. 3’ mistakes cause nascent duplex to open & exonuclease removes 3’ end, which is then resynthesized.
Spontaneous base loss
- Bases, usually purines, ‘fall off’ leaving an abasic site with an intact phosphodiester backbone. This can lead to doublestranded breaks
Deamination
- Removal of an amine group via hydrolysis
Oxidative stress
- Reactive oxygen species damage bases, leading to base loss, phosphodiester bond breaks, and ss DNA breaks
Ionizing radiation
- Radiation energy lyses water to create ROS –> oxidative stress
UV light
- UV energy covalently binds adjacent bases - usually thymine & this blocks transcription and replication
Intended mutagenesis
- Rearrangement of B & T cell receptors during development.
- Activation induced deaminase (AID) introduces point mutations
Adduct formation
- DNA adduct = segment of DNA bound to a chemical. Interferes with base pairing, transcription, and replication.
AMES test
Method of testing chemicals for carcinogenic nature.
Repair Pathways
Nucleotide Exicision Repair (NER)
Base Excision Repair (BER)
Mismatch Repair (MMR)
Non-Homologous Endjoining (NHEJ)
Homologous Recombination (HR)
Nucleotide Exision Repair
(NER)
Repairs UV-induced DNA damage.
UV damage binds neighboring bases (usually thymines). Creats distortion that recruits repair proteins.
DDB1/2 recognizes and binds damage.
XPB and XPD form complex with TF2H (helicase) unwinds area and endonuclease (XPG or ERCC1-XPF) cuts backbone.
Damaged segment is excised & degraded.
DNA polymerase fills in gap.
DNA ligase fuses gaps.
Base Excision Repair
(BER)
Removes damaged bases from DNA. Example: deamination, where cytosine is converted to uracil. There are specific DNA glycosylases that recognze specific incorrect bases and uses a ‘base flipping’ mechanism to remove the incorrect base.
DNA glycosylase excises the damaged base.
AP endonuclease (APE) forms nick and lyase removes sugar phosphate.
DNA polymerase fills gap. DNA ligase seals nick.
Mismatch Repair
(MMR)
Repairs mismatches from replication errors, heteroduplexes from recombination & deamination of cytosine to thymidine.
Prokaryotes use methylation to distinguish strands.
Eukaryotes might distinguish strands via processivity factor interaction or detection of nicks in new strand.
The parent strand is methylated. Daughter strand is unmethylated after replication.
MutS binds misatch base and ATP.
MutH recognizes methylated strand.
MutL and MutS function as homodimers. MutH cleaves DNA following MutL stimulation. Daughter strand is removed.
Gap filled in by DNA polymerase and ligase.
Daughter strand gets methylated
Non-homologous end-joining
(NHEJ)
Most common mechanism of repairing ds breaks in non-dividing cells or euchromatin.
Ends of DNA flanking dsbreak are rejoined. Results in deletion of DNA sequence originally present in the gap.
Ku70/Ku80 rapidly detects ds breaks.
DNA-PKcs bind & recruit Artemis endonuclease that ‘cleans up’ ends. DNA ligase fuses ends.
Homologous Recombination
(HR)
Stimulated by persistant ds breaks.
MreII (of MRN complex) makes initial DNA resection to provide 3’ DNA end for invading strand.
MRN (Mre11, Rad50, Nbs1) recruits CtIP, which causes extensive DNA resection.
BRCA1 recruits RPA, which coats ssDNA
ATR is activated. BRCA2 recruited, mediates RAD51 loading for strand invasion.
3 scenarios:
1) D-loop cleaved, get crossover
2) Holliday junction formed and resolved with no crossover or with crossover
3) Holliday junction formed & resolved by endonuclease. Dangerous!
Features of Hereditary Cancer
- high risk of developing cancer
- younger age of onset
- multiple primary cancers
- generally have family history
- less commin in general population than sporatic cancer
Knudson hypothesis
Two-hit theory.
Cells initate tumors when they have two mutant alleles. Persons who inherit one mutant allele must have second somatic mutation to initate tumorigenesis.
Example 1: Breast cancer
- BRCA1 & BRCA2 are tumor suppressors with autosomal dominmant inheritance. Gender related - females more likely to develop cancer than males
Example 2: Colorectal cancer
- Familial adenomatous polyposis (FAP) accounts for 1% of colorectal cancer cases. Caused by mutation to APC gene (tumor suppressor, neg regulator of B-catenin & interacts with E-cadherin
- Hereditary nonpolyposis colectral cancer (HNPCC) AKA Lynch syndrome accounts for 5% of colorectal cancer cases. Mutated genes belong to DNA mismatch repair (MMR) family.
- Autosomal dominant inheritance. Not gender inheritance
- Does not show multiple polyps in colorectal track
Microsatellite
Microsatellite = tract of reptitive DNA motif
Microsatellite instability: can determine if patient has DNA repair deficiency by assesing whether microsatellite regions have more or less copies of repeats.
- HNPCC is due to defects in MMR machinery. Can assess microsatellite instability.
Association Studies
Approach to link genetic risk factors to common cancers.
DNA from individuals sequenced –> variation at a single nucleotide identified –> some indivials will have one verson of the SNP, some the other –> SNP can be associated with diease
Chip/microarray: used to genotype certain regions
Genome-wide association study (GWAS): looks at up to 5 million SNPs. Creates Manhattan plot to find SNPs associated with phenotype.
BUT we don’t know which gene is associated with the SNP, we just know the relative location.
Strategies to identify causal gene and functional SNPs
Expression genetics-eQTL mapping
- mRNA levels measured to examine gene expression. SNP is correlated with expression levels. SNP can be cis or trans to a gene.
Chromosome confirmation capture (3C)
- technique to analyze organization of chromosomes in cells natural state
- handy for studying long distance interaction
ChIP sequencing
- maps protein-DNA interactions
- can map alterations in txn factor binidng in response to cancer state
- DNA + bound proteins fragmented and immunoprecipitated–> DNA released –> fragments sequenced –> sequnces mapped to genome & peaks identified
Complex Disease - Sporadic cases
- appears to cluster in families
- multiple genes, each with low penetrance
- segregation analysis can provide estimates of genetic and environmental contribution to disease
NGS & Cancer
NGS can provide a picture of the cancer genome by detecting major alterations.
Whole genome vs exome sequencing (sequences exons)
- WGS can detect SNPs and copy number variation
- WES is cheaper than WGS
- WES reflects only transcribed protein products
- WES has higher coverage than WGS
Somatic abnormality databases
COSMIC = catalogue of somatic mutations in cancer
TCGA = the cancer genome atals
UCSC cancer browser
Cancer targeted therapy vs personalized cancer therapy
Cancer targetd therapy are ineffective and will ultimately fail because they do not account for tumor heterogeneity.
Liquid biopsy
Liquid biopsy fractionates whole blood into mononuclear cell fractions and plasma/serum. From these, DNA, RNA, and proteins can be extracted from cirulating tumor cell OR from free-floating DNA/RNA, exosomes, and platelets.
From this, circulating tumor DNA can be correlated with tumor burden, allowing for early detection and genetic profiling of the tumor.
Definition and characteristics of benign and malignant tumors
Benign:
- cells closely resemble, and may function, like normal cells
- stay localized to appropriate tissue
- small in size
- fibrous capsule delineates the boundaries of the tumor
- causes problems if bulk intereferes with normal function, or if they secrete excess biologically active substances
Malignant:
- express some proteins characteristic of tissue
- grow and divide more rapidly than normal
- most invade surrounding tissue, enter cirulatory system, and proliferate at sites away from origin (metastasis)
- loss of physical barriers
- most diagnositic property is presence of invasiveness
- less well-differentaited than normal cells or benign tumors
- require angiogenesis (new blood vessels) to grow
Major difference of malignant tumors is their invasiveness and spreading.
Aquired functional capabilities of cancer cells
-emerging hallmarks
-enabling characteristics
Functional capabilities
- evading apoptosis - p53
- self-sufficiency in growth siganls - HRas
- insensitivity to anti-growth signals - pRb
- tissue invasion & metastasis - VEGF
- limitless replicative potential - hTERT
- sustained angiogenesis - E-cad
Emerging hallmarks
- deregulating cellular energetics
- avoiding immune destruction
Enabling characteristics
- gene instability and mutation
- inflammation
Genetic instability and clonal selection
-multi hit model for carcinogenesis
A single mutation is note enough to cause cancer.
Multi-hit model of cancer: succession of individual mutations, each of which confers a slight growth adventage, is necessary for cancer to develop.
Clonal evolution: tumors develop through multiple rounds of mutation and proliferation. Each step, a single cell mutates in a way that enhances poliferation. Progeny of mutant cells become dominant clones in tumor.
Cellular senescence versus immortality
Cell proliferation in culture are able to initially proliferate before entering into senescence, where they are viable but non-proliferating. Senescent cells have degraded telomeres.
Cells that can escape senescence enter ‘crisis’, where they continue proliferating. Telomerase is reactivated at crisis level. Very few cells escape crisis and become immortal.
Genetic control of cell proliferation
-regulation of cell population size
-oncogenes vs tumor suppressors
-mechanisms to generate oncogenic mutations
7 protein classes involved in controlling cellular proliferation
- signaling molecules
- signaling receptors - intracellular & extracellular
- intracellular transducers
- transcription factors
- DNA-repair proteins
- cell-cycle control proteins
- apoptotic proteins
Regulation of cell population size
Regulated by positive and negative inputs that control cell number. Caspase is a key effector molecule of apoptosis. Tumorigenesis can result from increased cell division or decreased apoptosis
Proto-oncogenes: normal cellular genes whose products are involved in cellular growth control pathways. Activation of a proto-oncogene to an oncogene usually involves a gain-of-function mutation that acts dominantly.
Conversion to oncogenes: At least four mechanisms
- point mutants = consitutively active proteins
- local gene amplification leading to overexpression of a proto-oncogene
- chromosomal translocation that juxtaposes a growth regulatory gene with a different promoter that results in misexpression of the gene
- chromsomal translocation that fuses two genes to make a chimeric protein whose activity is unlike the parental proteins and often constitutive
Definition of stem cell
-properties and types of stem cells
-clonal succession and expansion of stem cells
-cancer stem cells and anticancer drugs
Stem cell: cells that have the capacity to divide and replenish themselves while retaining the ability to differentiate into specialized cell types
Properties:
- self renewal
- potency
Totipotent SC: can be differentiated into any cell type including extra-embryonic
Pluripotent SC: derived from the inner cell mass of the blastocyst, can be differentiated into all three germ layers (ectoderm, mesoderm, and endoderm), cannot create extra-embryonic tissue
Multipotent SC: can form a limited number of differentaited cell types
Unipotent SC: can differentiate into only one cell type but are capable of self renewal
