DNA & Genome Structure Flashcards
1
Q
Eukaryotic DNA structure - recap
A
- Hierarchical:
o Double helix DNA slit into 2 strands w/genetic information
o Coiled up into double helix →-ve backbone interacts w/ +ve histones
o Histones coil up into coils → form coiled coils → form supercoils - Huge amount of DNA into v. small space
2
Q
Bacterial DNA structure -recap
A
- Less coiled than eukaryotic
- Highly organised supercoiled circular nucleoids
- Some circular; some linear
- Have scaffolding proteins e.g. Histone-like nucleoid structuring protein (H-NS)
3
Q
DNA synthesis – recap
A
- Synthesis of polynucleotide chain by DNA polymerase
o Requires ATP hydrolysis
o Mg2+ - Nucleophilic attack by 3’-OH on growing chain → hydrolyses incoming base → form phosphodiester bonds
- Polymerase always works 5’ –> 3’ → DNA strands antiparallel
- Endonuclease = cut into DNA → makes nick
- Exonuclease = digest DNA from open ends
4
Q
Stability of DNA
A
- DNA carries genetic information from one generation to next → must be stable over many lifetimes
- DNA is susceptible to damage from environmental mutagens (e.g. smoke, chemicals, radiation)
o ~10,000x per cell per day
o Leads to disease e.g. cancer
5
Q
DNA Damage
A
- Can block replication/transcription
- Can cause alteration in genetic code (mutation)
Causes:
1. Chemical alteration to DNA
o Exogenous – environmental mutants e.g. UV radiation
o Endogenous – internally generated damaging agents e.g. hydroxyl radicals
2. Spontaneous damage to DNA
o e.g. deamination, depurination
6
Q
Examples of DNA damage induced by exogenous agents
A
- UV light causes pyrimidine dimers (most common: T-T)
o 2 adjacent pyrimidines covalently joined by cyclobutene ring structure
o Can lead to skin cancer - Alkylation in wrong place of DNA
o Alkylation = addition of methyl/ethyl groups to various positions on DNA bases
o E.g. alkylation of the O6 position of guanine → O6-methylguanine → changes Watson-Crick base paring potential of guanine - Carcinogens (e.g. benzo-pyrenes)
o Reactive bulky groups added to various positions within DNA bases
o Often have to be activated by cellular enzymes e.g. cytochrome P450 (catabolic enzymes) try to break down foreign bulky chemicals but create reactive intermediates then react w/DNA
o Cytochrome P450 highly relevant for drug metabolism → they break down into many products for foreign chemicals like drugs
o Liver packed with cytochrome P450
o Bulky groups prevent DNA polymerase moving properly through DNA
o Can potentially change Watson-Crick base pairing potential → when DNA replicates will incorporate wrong base
7
Q
Spontaneous damage to DNA
A
- Deamination of adenine, cytosine and guanine
o Taking off amine off DNA base
o E.g. NH2 of cytosine replaced by O → uracil (same base pair potential)
o E.g. NH2 of adenine replaced by O → hypoxanthine (different base pair potential)
o Hypoxanthine has C=O and NH that can H-bond (looks like G) → has H-bond acceptor which forms 2 H-bonds w/cytosine
o Just losing one amine group spontaneously → DNA replicates telling cell to add C instead of T at position of A → creates permanent mutation in DNA → transfers down through generations - Depurination
o Removing purine (A and G) → cleaving bond between purine and deoxyribose
o Forms apurinic (AP) site in DNA → replaced entire DNA base w/OH → DNA loses entire coding potential → information lost from the cell
o “Silver soup tureen → purine → AG”
8
Q
2 general types of DNA repair mechanisms
A
- Direct reversal
o Chemical reaction responsible for DNA damage is reversed
o Usually needs specific enzymes for specific kinds of DNA damage
o More specialised repair mechanism
Example: Repair of pyrimidine dimers caused by UV exposure
o Photoreactivation → uses photo-reactivating/photo-lyase enzyme
o Uses visible light to break cyclobutene ring
o Enzymes found in bacteria, yeasts, some plants/animals but not in humans - Excision repair
o Remove DNA damage → single bases or stretches of DNA → replace w/new synth DNA
o More common and most important in humans than direct reversal
Example: alkylation → methylation of guanine → O6-methylguanine base pairs with thymine
o Repaired by enzyme: O6-methylguanine methyltransferase
o Has reactive cysteine in active site → reacts w/methyl group to form cov. bond (S-CH3)
o Reaction is not fully enzymatic → cell needs to reduce S-CH3 back to Cys to be reused
o Common enzyme because widespread in prokaryotes and eukaryotes
9
Q
Mechanisms of Excision Repair
A
- Base-excision repair
o Base removed leaving deoxyribose backbone intact - Nucleotide-excision repair
o Nucleotide removed → gap in one strand (oligonucleotide is usually removed) - Mismatch repair
o Repair of post-replicative mismatches
o After DNA synthesis → repairs errors of DNA Polymerase
10
Q
Base-excision repair
A
- Start w/DNA w/lesion e.g. G:U → no H-bond correctly → bulge in DNA (U by deamination of C)
- Cell detects lesion using uracil DNA glycosylase → recognizes uracil
- Glycosylase cleaves uracil-deoxyribose bond → leaves AP site → gap in DNA
- AP endonuclease recognizes AP site → cleaves DNA chain hydrolysing phosphodiester backbone → remaining deoxyribose removed by deoxyribose-phosphodiesterase
- Resulting gap filled by DNA Polymerase → sealed by ligase → incorporates C opposite G
11
Q
Nucleotide excision repair (NER)
A
- Process discovered because of disease such as…
- Xeroderma Pigmentosum → genes involved in repair pathway are damaged → T-T cannot be removed from cells quickly → cannot repair UV damage → damage does heal
- NER is major mechanism to repair thymidine dimers in humans
- Bulge in DNA recognised and cleaved on both sides of T-T dimer by 3’ and 5’ endonucleases
- Helicase unwinds DNA resulting in excision of oligonucleotide containing damaged bases
- Resulting gap filled by DNA polymerase from 5’ –> 3’ → sealed by ligase
o In E. coli DNA Pol 1; in humans DNA Pol ß - In E. coli:
o Catalysed by 3 gene products (uvrA, B, C)
o Mutations of genes leads to high sensitivity to UV
o UvrA recognises damaged DNA
o UvrB/UvrC endonucleases cleave at 3’ and 5’ sides; helicase activity excises oligonucleotide - In eukaryotes:
o Catalysed by RAD (radiation damage) gene products in yeast
o Genes identified in humans with Xeroderma Pigmentosum (rare genetic disease affecting ¼ mill people)
o 7 different repair genes involved → v. highly conserved → important in maintaining genomic integrity because of all the time exposed to UV
12
Q
Mismatch repair in E. coli
A
- Mismatch repair system detects and excises mismatched bases in newly replicated DNA
- Must distinguish parental strand from newly synthesised daughter strand
- DNA in E. coli is methylated by Dam methylase
- Following replication new daughter strand will not be methylated at dam sites → DNA is hemi-methylated
- Methylases also protect bacteria from own restriction enzymes
o Foreign DNA from phage viruses infect bacteria → not methylated → cut up by restriction endonucleases → methylated DNA not cut - Once cell knows there’s old strand and new strand because of hemi-methylated DNA → mutHLS repair system removes lesion on new strand
o MutS recognizes mismatch
o MutL inly binds MutS at mismatches (ATP hydrolysis, forms DNA loops, translocates along DNA looking for hemi-methylated dam site) → either remains bound to mismatches or migrates away translocating in both directions
o MutH endonuclease activated when bound to MutL → cleaves unmodified strand opposite site of hemi-methylation (GATC) → can discriminate newly synthesised DNA → discrimination does not require complex to be bound at mismatch site (presence of complex enough to signal mismatch is present)
o Random process → mistake can happen anywhere
o Depending on orientation nick can be upstream/downstream of mismatch
o Different exonucleases required depending on polarity (have different 5’/3’ ends)
o In one direction use exonuclease 7 (recJ); other direction use exonuclease 1 → end result is the same regardless of orientation but exonuclease digests single-stranded DNA until past lesion sites so lesion removed in new DNA → old strand still contains correct DNA base → can use DNA Pol and ligases to fill in gap and seal final nick by ligating P to 3’-OH
o Extra components (UvrD helicase) to dissociate strands
13
Q
Mismatch repair in mammals
A
- Recognition mechanism based on fact that DNA in eukaryotes has lots of strand breaks → DNA replication is semi-discontinuous
o Cell differentiates between old DNA (no gaps) and new DNA (gaps) - Eukaryotic DNA contains many replicons that occur in different strands → end up w/Okazaki fragments on both strands
- Cell can consistently detect mistakes in new DNA
- Analogous to E. coli mismatch repair
o Instead of MutHLS system → MHS complex - Method:
o Lesion in DNA caused by newly-replicated DNA
o Enzyme complex recognises old and new strand and binds in particular orientation
o Helicase and exonuclease nicking either side of the lesion
o Exonuclease digests oligonucleotide stretch
o DNA polymerase fills gap and DNA ligase seals gap - In humans, mutations in hMsh2 and hMsh1 genes are cause of inherited non-polyposis colorectal cancer:
o Affects 1:200
o Causes ~15% of UK colorectal cancers
14
Q
Double strand break
A
- DNA broken to leave 2 free open stranded ends
- Can occur due to:
o DNA damage
o DNA lesions prevent processivity of DNA Polymerase during DNA synthesis
o DNA nicks by endonuclease damage
o Ionising radiation (e.g. X-ray damage)
15
Q
Non-homologous end joining (NHEJ)
A
- Simplest mechanism; found in eukaryotes
- Take broken ends ligated by enzymes (5’P joined to 3’OH)
- Error prone process → often introduces mutations into DNA
o Complicated because sometimes can have unwinding/overlapping at broken ends → deletion or addition base pairs
o If you have microhomology, can predict indels within DNA as consequence - Exploited by modern genome editing tech (e.g. CRISPR/Cas9) for targeted mutations/knockouts
o When guiding CRISPR/Cas9 to make ds break in genome, depending on chosen sequence at the ends, can increase/decrease favourability of having NHEJ or introduction if indels
o Useful because if pick sequence likely to have microhomology and lead to deletion → v. efficient way of knocking out target gene on genome by changing reading frame, introducing stop codons, etc - Associated with recombination of variable regions in antibodies within human immune system
o Generates more diversity within antibody regions - Only found in certain bacteria → homologous to human machinery
o Kill microplasma bacterium by cutting genome because cannot repair properly by NHEJ
16
Q
Homologous recombination
A
- General mechanism for repair where intramolecular template information has been lost
o Uses information from paired DNA to repair broken strands → stimulated by ds breaks
o Also repairs nicked DNA → not always fully ds breaks - Relative error-free process
- Requires homologous DNA as template
o In diploid organisms: pairs of chromosomes → every gene has 2 alleles → natural source of homologous DNA
o In haploid organisms: homologous DNA template readily found at replication forks - When making ds breaks in biotech can provide own homologous template → can have mutations as long as ends are 100% homologous
o Exploited by modern genome editing technology (e.g. CRISPR/Cas9) to make site-targeted gene repair, integration or modification - Basic process:
1. Break two homologous DNA pairs
2. Pair strands by Watson-Crick base pairing
3. Reform phosphodiester bonds so new strands are crossed over and joined together
4. Break the strands and reform again
5. Any gaps filled in by DNA polymerase and ligase - Intermolecular HR:
o Double crossover events common in meiosis
o Generate diversity in eggs/sperms for gene swapping over
o Source of variation for natural selection and evolution in longer term - Intramolecular HR:
o Small stretches of DNA recombine between themselves within same stretch of DNA
o Direct repeats → same orientation → cuts out DNA from longer stretch and join what was on either side of that region
o Inverted repeats → pointing towards each other → after recombination, flip orientation of DNA stretch within a longer region
o Evolution: can change orientation of regulatory promoter regions, enhancers, genes, etc thus change overall gene expression pattern of a cell
17
Q
E. coli RecBCD recombination pathway
A
- Provides model for initial steps of homologous recombination
- Orientation of DNA 3’-OH and 5’-P ends → geometrical constraint
- Exploited by bacterial genome editing technology with oligonucleotides → “recombineering”
- Ds break in DNA → damage causes initiation of HR
- RecBCD uwinds DNA and degrades one strand exposing 3’ strand
- 3’ strand bound by RecA to form a filament → stabilise it and allow strand invasion
- Ss RecA filament invades homologous strand and pairs up
- RecA comes off → start of crossover
- Have a d-loop in homologous strand (bulge of unpaired ssDNA)
- To complete formation of crossover → break second DNA → nicking
- End up w/ fully crossed over DNA → strand exchange of homologue
- Gaps filled by DNA polymerase
- Once reaches 5’-end → ligation to repair phosphodiester backbone
- DNA strands connected by covalent linkage through the molecule and crossed over in the middle (holliday junction)
- Resolution: get rid of crossover → break DNA and ligate again → final resolved products
18
Q
Homology search and strand invasion
A
- Precise details of homology search not known
- RecA filament formation is essential for strand invasion
- Helical nature of RecA filament can form triplex structure w/homologus DNA duplex
o Start w/ ssDNA coated by RecA protein → 3 strands of DNA come together during invasion: invading ss and 2 strands unwound to form d-loop
o One original DNA strand displaced by invading strand - ATP required to drive process over longer lengths of DNA
19
Q
Holiday junction and recombination
A
- Found by Robin Holliday in 1964
- Following strand invasion, invaded duplex must be nicked using specific nucleases
- Gaps on strand must be resealed using ligase
- Holliday junction forms:
o Drives swap of genetic information by moving randomly using RuvA+RuvB
o Carries part of one chromosome/DNA template along w/them
o Depending on how much it moves, determines amount of genetic information swapped - RuvA forms flat structure which protects crossover and stabilises it
o Contains hydrophobic ‘pin’ in the middle helps separate strands → machinery slides along and swaps DNA - 2x RuvB motors bind either side of RuvA and use ATP to translocate DNA
- KEY: Homologous strands are similar but do not have to be identical
