Genome Integrity
Terms
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- Topoisomerases
- •Catalyze transient breakage and reunion of DNA strands.
- XP-V (variant)
- •A defect in translesion synthesis •Same presentation as XP •Normal capacity to repair damaged DNA by NER •UV sensitivity is due to defect in specialized DNA polymerase (pol h (eta)) that mediates translesion synthesis •Pol h has evolved to incorporate AA opposite cyclobutane thymine dimer •In its absence, lesions that have not been repaired can not be replicated and DNA replication stalls, which is a lethal event
- Hexameric replicative helicase
- ATP-dependent unidirectional translocation on single-stranded DNA with concomitant displacement of the opposite strand results in DNA unwinding
- Topoisomerase inhibitors
- Used as both antibiotics (e.g. ciprofloxacin) and anti-cancer drugs (e.g. etoposide)
- Lagging Strand
- •Strand that is synthesized dis-continuously. •Okazaki fragments are ligated by ligase •Primed de novo per okazaki fragment •Goes 3' -> 5'
- The "unwinding" problem
- •Replication fork progression introduces +ve supercoiling (overwinding) ahead of the fork. •Other genome rearrangements (e.g. compaction of DNA into chromatin) also introduce torsional stress.
- Torsional Stress
- •Replication fork progression introduces +ve supercoiling (overwinding) ahead of the fork. •Compaction of DNA into chromatin
- SSB
- Cooperative protein binding of monomers straighten region of single stranded chain that may have gone to secondary structure.
- Mismatch repair
- •Post replication or recombination •Recognition of mismatch (mispaired base, insertion or deletion) •Incision of newly synthesized strand •Exonucleolytic digestion beyond site of mispair •Fill-in and ligation
- Spontaneous changes in DNA chemistry
- •Deamination •Depurination, results in loss of a base, i.e. an abasic site •Oxidative damage from reactive oxygen species
- Type II topoisomerases
- Cut and ligate double strand cuts
- Somatic Cell
- Reduced (TTAGGG)n limited replication
- Telomeres
- Protect ends of chromosomes from fusion and degradation
- UV radiation
- •Py - Py 6-4 photoproduct •Cyclobutane pyrimidine dimer
- Base excision repair
- Recognition of unusual bases in DNA, e.g. U -> Removal of base(uracil-N-glycosylase) -> Incision of P'diester backbone -> Removal of "abasic residue" -> Fill-in and ligation
- Type I topoisomerases
- Cut and ligate single chain cuts
- DNA Ligase
- Restores the continuity of the phosphodiester backbone by covalently linking 3' OH to 5' phosphate ends in an ATP-dependent reaction
- Nucleotide excision repair (XP A-G)
- •Recognition of structural alteration in DNA •Incision of P'diester backbone on both sides of lesion •Removal of damaged oligonucleotide •Fill-in and ligation
- Topoisomerases
- Catalyze transient breakage and reunion of DNA strands relieving torsional stress.
- Cancer Cell
- Up to 55kb of TTAGGG. Persistent growth but also chromosome instability.
- Senescent Cell
- Telmeric ends with no TTAGGGn. Breaking-fusion-bridge cycle, Chormosome instability, and Cell Death
- Homologous recombination
- A mechanism to restart replication at sites of collapsed replication forks without loss of DNA sequence
- Spontaneous Genotoxicity
- •DNA replication errors •Spontaneous changes in DNA chemistry
- Primer
- •A small stretch of RNA used to start replication. •Synthesized by primase.
- Eukaryotic cell cycle
- •G1 -> S -> G2 -> M -> G1 •DNA replication occurs during S phase •Repair synthesis may occur in G1 and G2
- Chemicals
- •Alkylating agents, e.g. EMS •Cross-linking agents, e.g. cisplatin •Metabolic activation, e.g. aflatoxin
- Telomerase
- •A ribonucleoprotein that adds telomeric repeats onto the ends of chromosome in some cells (e.g. gametes) to prevent shortening. •Because of the polarity of DNA synthesis telomeres are shortened during each division cycle in most somatic cells.
- The "unwinding" problem
- •Replication fork progression introduces +ve supercoiling (overwinding) ahead of the fork •Other genome rearrangements (e.g. compaction of DNA into chromatin) also introduce torsional stress
- Type I topoisomerases
- Cut and ligate single chain cuts
- Environmental DNA damage
- •Ionizing radiation (e.g. X-rays) •UV radiation •Chemicals
- Genes implicated in HNPCC
- •MLH1 •MSH2 •MSH6 •PMS2
- Double Strand Break
- When a nick prematurely ends replication
- Hereditary nonpolyposis colon cancer (HNPCC) aka Lynch syndrome
- •A patient presents with constipation, rectal bleeding, abdominal discomfort and unexplained weight loss •The doctor orders a colonoscopy which detects polyps that are biopsied. Histopathology is consistent with adenocarcinoma •There is a family history of early onset colon and other GI cancers •PCR testing reveals microsatellite length polymorphism that is indicative of microsatellite instability.
- DNA Replication Fork
- Leading and lagging strand synthesis is coupled and both polymerases move in the same net direction
- Non homologous end joining (NHEJ)
- •"Simple" ligation of broken DNA ends after some end trimming •Involves some loss of DNA sequence
- Proofreading
- Increases the fidelity of DNA synthesis ~100 fold (100,000 errors/genome) by excising misincorporated nucleotides
- UV Damage
- Cyclobutane thymine dimer and 6-4 photoproduct
- DNA Polymerase mechanism
- •3' hydroxyl acts as primer •Incorporates dNMP at 3' end (OH) •Chain growth is 5'-3' •Nucleotide selectivity of polymerase has a fidelity of ~ 1 error in 1x105 bases (10 x 106 errors/genome)
- Ionizing radiation (e.g. X-rays)
- •Direct •Indirect (reactive radicals, i.e. oxidative damage)
- Functional elements of chromosomes
- •Replication origins •Centromeres •Telomeres
- DNA Clamp (PCNA)
- Prevents dissociation of DNA polymerase during replication. Loaded onto DNA via a Clamp Loader(RFC-3)
- Semi-Conservative Mechanism of DNA Replication
- •One parental strand acts as a template for the newly synthesized strand in the new double helix. •Human DNA is constructed this way.
- Frequency of mutations in HNPCC families
- •MLH1 and MSH2 account for 90% of mutations in HNPCC families. •MSH6 7-10% of mutations in HNPCC families •PMS2 <5% of mutations in HNPCC families
- Topoisomerase inhibitors
- Used as both antibiotics (e.g. ciprofloxacin) and anti-cancer drugs (e.g. etoposide)
- Origins of Replication
- Specific DNA sites that are recognized by sequence specific DNA binding protein that mediate localized unwinding of the DNA duplex
- Xeroderma Pigmentosum
- •Multifactorial disease •Extreme sensitivity to sunlight •Blistering or freckling on minimum sun exposure •History of skin cancer •Neurological abnormalities (mental retardation and hearing loss) • Inherited as autosomal recessive •Several complementation groups (A-G and V = Variant) •Rare in US (1:1,000,000) more common in Japan (1:100,000) •Defect in nucleotide excision repair
- Centromeres
- Assembly point for the kinetochore
- DNA Polymerase
- •Looks like right hand •Template Dependent •Primer-Dependent •Utilizes deoxynucleotide triphophate precusors•
- Type II topoisomerases
- Cut and ligate double strand cuts
- Embryonic Stem Cell
- 3-20kb of (TTAGGG)n allowing indefinite replication
- Chromosomal DNA Replication
- •Replication is bidirectional from multiple origins
- Telomeres
- Ends of chromosomes with TTAGGG repeat that are replicated by a specialized polymerase
- Fidelity
- ~ 1 error in 1x10¹⁰ bases
- Conservative Mechanism of DNA Replication
- Both parental strands act as templates for two newly synthesized strands that associate into their own helix.
- Leading Strand
- •Strand that is synthesized continuously •Primed once •Goes 5' -> 3'
- DNA replication errors
- •Mis-incorporation during and post-replication •Strand slippage