Flashcards in Organisation, Replication & Repair of Genomes Deck (69):
What are the functions of DNA?
1. Storage of genetic information: Ensures vast amounts of DNA can be stored in a compact space (e.g. nucleus).
2. Replication of genetic information: Double-stranded helix means that DNA can be replicated easily through semi-conservative replication.
3. Interpretation of genetic information: Provides simple way for genetic information to be read during gene expression, as well as control over that reading.
What is the structural significance of the pentose sugar in nucleic acid?
Usually, 4 carbons are coplainer while 1 is above/below plane of the ring. For B-DNA, carbon 2 is out of the plane.
What are the structures of nitrogenous bases?
- All nitrogenous bases are based around an aromatic ring with different groups attached.
- Purines (A, G) are bigger and contain 2 rings in their structures.
- Pyrimidines (C, T, U) are smaller and contain 1 ring in their structures.
What is the difference between a nucleoside and a nucleotide?
- Base + Sugar = Nucleoside
- Base + Sugar + n phosphate(s) = Nucleotide
What is the significance of the structure of a polynucleotide?
The polynucleotide strand is polar (from 3'-5' direction). This is essential in DNA replication and translation.
What are the differences between ribose and deoxyribose?
- Deoxyribose has a -H in place of an -OH group on 2' carbon of ribose.
- Ribose is more polar than deoxyribose, and so is more hydrophilic.
- This allows RNA to exist in single-stranded form more easily but also makes RNA less stable and more vulnerable to hydrolysis.
What is the gross structure of a DNA molecule?
DNA consists of 2 antiparallel polynucleotide strands, held together by hydrogen bonding between the nitrogenous bases, in a double right-handed helix configuration.
What is Chargaff's rule?
Chargaff's rule states that:
- %A = %T
- %C = %G
What are the structural properties of the DNA helix?
- Right-handed helix
- 10.5 bp/turn
- Raises by 34Å per turn
What are the 3 types of DNA?
- A-DNA: Formed when B-DNA is dehydrated. Has deeper major grooves and shallower minor gorrves.
- B-DNA: Most common form of DNA.
- Z-DNA: Left-handed DNA helix formed in the presence of specific sequences (e.g. GC repeat). They may be involved in transcription.
What factors does the process of DNA melting depend on?
2. Length of DNA
3. Base sequence of DNA
3. Ionic composition of solution
What are the problems with storing DNA?
1. -ve charge: Attracts +ve proteins, which may disrupt the structure of DNA.
2. Long: Difficult to fit into the compact space of a nucleus or prokaryote.
What are the solutions to packaging DNA?
What mediates supercoiling?
Topoisomerases mediate supercoiling. There are 2 types of topoisomerases:
1. Type I topoisomerase: Mediates supercoiling by mechanisms involving cutting one strand of double-stranded DNA.
2. Type II topoisomerase: Mediates supercoiling by mechanisms involving cutting both strands of double-stranded DNA.
What are the clinical significances of topoisomerases?
- Topoisomerases are vital in the maintaining DNA.
- Novobiocin, a type II topoisomerase inhibitor, is used as an antibiotic.
- Doxorubicin, a type II topoisomerase inhibitor, is used as a chemotherapeutic.
What is a nucleosome?
- A nucleosome is a DNA-histone complex.
- The structure contains a histone barrel, consisting of an octomer of H2A, H2B, H3 and H4.
- 140-150 bp long strands of DNA are wrapped around the histone barrel ~1.6 times.
- Each histone barrel has a N-terminal and C-terminal tails that can be modified to control DNA packaging.
What is chromatin?
- Nucleosomes are linked together by linker DNA to form a 10 nm chromatin fibre.
- H1 causes 10 nm fibre to further coil into 30 nm chromatin fibres.
- 30 nm chromatin fibres are threaded through scaffolding proteins and further condense to from the visible chromosome structures visible in mitosis.
What are the functions of DNA packaging?
1. Allows DNA to fit into the small space of the nucleus.
2. Allows gene expression to be controlled.
How can DNA packaging be controlled?
- Modifications of the histone tail affect the way chromatin is packaged. For example, acetylation of DNA cancels the +ve charge on lysine residues, which reduces the attraction between histones and -ve DNA, reducing how tightly DNA is bound in nucleosomes.
- Remodelling proteins are capable of changing the sections of DNA packaged into nucleosomes as well as the density of nucleosomes.
How can DNA contribute to disease?
1. Genetic: Mutations can be passed from parent to offspring to cause genetic dieases (e.g. CF).
2. Spontaneous mutations: Excessive DNA damage and/or malfunctioning DNA repair mechanisms can lead to spontaneous mutations that cause diseases (e.g. cancer).
Who carried out the experiment to prove that DNA carried out semi-conservative replication?
Meselson & Stahl
What is the name given to the initiation site of DNA replication in prokaryotes?
What is the process of initiation of DNA replication?
1. Multiple DnaA molecules bind to OriC, causing a small strand of DNA to unwind.
2. DNA helicase (DnaB) is loaded onto on ssDNA strand in this segment of unwound DNA with the help of DnaC.
3. Helicase continues to unwind the dsDNA into 2 ssDNA strands, moving replication fork forwards.
What is the structure and function of DnaB?
- DnaB is a 6-subunit ring structure with a central pore through which DNA is threaded.
- It functions on the basis of steric exclusion whereby the pore allows one strand of DNA through but displaces the other so that DNA unwinds.
What are the functions of SSB?
1. To prevent DNA from rewinding
2. To prevent ssDNA from being degraded by the cell
What is the main DNA polymerase in prokaryote DNA replication?
DNA Pol III
What are the requirements for DNA polymerase to function?
1. Template strand
2. Pre-existing 3' -OH
3. free dNTPs
What state is DNA polymerase III usually found in?
In a complex of 2-3 DNA Pol IIIs called Pol III holoenzyme which simultaneously replicates leading and lagging strand.
What determines proofreading capability of DNA Pol III?
3'-5' exonuclease activity
What is the main function of DNA Pol I?
To excise primers
How does lagging strand replication occur?
- In fragments called Okazaki fragments.
- Both leading and lagging strands are replicated in the same direction by same holoenzyme, so trombone model used to flip lagging strand into the 3'-5' direction required by the polymerase.
How is processive replication achieved in leading and lagging strand?
- DNA Pol III drops off after adding 12 bp.
- β clamp used to ensure that Pol III continues to synthesise DNA without dropping off (processive).
- β clamp added to holoenzyme complex with help of clamp loader protein.
What are the differences between prokaryotic and eukaryotic DNA replication?
1. Prokaryotes only have 1 origin of replication while eukaryotes have many.
2. Eukaryotes have DNA & RNA in primers while prokaryotes only have RNA.
3. Eukaryote DNA replication regulated by cell cycle.
What is the process of eukaryotic primer excision?
1. RNAse H1 excises RNA portion of primer.
2. DNA polymerase activity displaces DNA portion to create flap.
3. Flap is excised by flap endonuclease.
What are clinical examples of topoisomerase inhibitors in cancer therapeutics?
- Camptothecin is topoisomerase I inhibitor
- Etoposide/doxorubicin are topoisomerase II inhibitors
What are the functions of telomeres?
1. Replication: To prevent chromosomes shortening after DNA replication
2. Capping: To prevent free DNA from undergoing recombination and fusion with other chromosomes
What is the sequence of human telomeres?
What is the functional architecture of telomerase?
- Telomerase is ribonucleoprotein consisting of RNA and protein components.
- RNA component acts as template for telomere synthesis.
- Protein component contains reverse transcriptase domain used to convert RNA template to DNA telomeres.
What is the limit of cellular division?
What are the uses of NGS (Next Generation Sequencing)?
- Allows genomes of different species to be sequenced quickly to determine evolutionary relationships.
- Allows genomes of individuals to be sequenced quickly to determine any predisposition to genetic diseases.
- Allows new pathogens to be identified.
What are the 4 classes of junk DNA?
- Simple sequence repeats
- Segmental duplications
- Transposon-derived repeats
- Processed pseudogenes
What are the 2 types of mobile genetic elements?
2. Retrotransposons (~40 human genome)
What are the 4 classes of MGEs?
1. Long interspersed nuclear elements (LINEs)
2. Short interspersed nuclear elements (SINEs)
3. Retrovirus-like elements
4. DNA transposon fossil
What are the functions of MGEs?
- Allows beneficial genes to be passed between bacteria.
- Facilitates mutations in evolution.
What are the important functions of reverse transcriptases?
- Telomerase activity
What are the conditions that favour DNA melting?
- High temperatures
- Low ionic content of solvent
What are the uses of DNA hybridisation?
- DNA probes (e.g. FISH)
- Southern blotting
What are the uses of restriction endonucleases?
- DNA splicing
- Restriction mapping
- Detecting genetic variation
What is the process of PCR?
1. dsDNA fragment to be replicated is added, along with primers (in excess), Taq polymerase and free dNTP.
2. Mixture heated to around 95o, melting the dsDNA to produce template ssDNA.
3. Mixture cooled to 55o (determined by primer composition, A/T = 2oC, G/C = 3oC, meaning that both primers should be similar in length/composition in order for their annealing temperature to be similar) to encourage primer annealing to ssDNA.
4. Mixture reheated to 72o to stimulate polymerase action and replicate template strands.
5. Cycle is repeated, with newly synthesised strands from previous cycle being used as templates. This accounts for exponential DNA replication.
What are the endogenous sources of DNA damage?
- Reactive oxygen species
- Reactive chemicals
- Chemical instability
What are the exogenous sources of DNA damage?
- Chemical mutagens
- UV radiation
- Ionising radiation
What are the types of DNA damage?
- Base alterations
- Sugar-phosphate backbone modifications
- Sugar-phosphate backbone breakage
What is the Ames test?
Uses salmonella to determine mutagenic properties of a chemical compound.
What are the types of DNA replication errors?
- Point mutations
- Base mismatch
- Incomplete replication
What are the consequences of faulty DNA repair mechanisms?
- Cell death
- Neurodegenerative disorders
- Developmental disorders
What are the types of single-strand repair mechanisms?
1. Direct damage reversal
2. Base excision repair
3. Nucleotide excision repair
4. Mismatch repair
What is the enzyme involved in direct damage reversal?
- DNA photolyase
What are the enzymes involved in base excision repair?
1. (Uracil) DNA glycosylase
2. AP endonuclease
3. DNA polymerase β
What is the clinical significance of base excision repair?
Mutations in UNG (glycosylase gene) is associated with glyoblastomas.
What are the proteins involved with nucleotide excision repair?
1. DDB domain
2. UvrABC complex
3. DNA polymerase
4. DNA ligase
What are the proteins involved with mismatch repair?
1. MutS & MutL
3. UvrD helicase
4. DNA polymerase
What are the human equivalents of MutS and MutL?
MutS = MSH2
MutL = MLH1
What is the clinical significance of NERs?
Mutations in NER enzyme genes results in xeroderma pigmentosa.
What is the clinical significance of mismatch repair?
Mutations in mismatch repair genes result in hereditary non-polyposis colorectal cancers (HNPCCs).
What are the proteins involved in NHEJ repairs?
- DNA polymerase
- DNA ligase 4 (LIG4)
What is the clinical significance of NHEJ repairs?
- Glyoblastomas are resistant to radiotherapy due to hyperactive NHEJ machinery.
- LIG 4 mutations result in LIG 4 syndrome which results in leukaemia and immunodeficiency.
What is the protein involve in HR repairs?
What is catenation?
- Supercoiling induced when 2 replications forks collide with each other.
- This is resolved by type II topoisomerases.