DNA Structure, Storage, Replication And Repair - Done Flashcards
How many base pairs per DNA helical turn?
10 - 10.5
How wide are the minor and major groves in DNA respectively?
Minor groove - 1.2nm wide
Major groove - 2.2nm wide
What’s the significance of the major groove?
The edges of bases are more accessible and lots of enzymes use this as a binding site - transcription factors
Also a target for medicinal chemists to exploit
What is the mechanisms of intercalating drugs on DNA?
Planar (aromatic or heteroaromatic) systems slip between the layers of nuclei acid pairs and disrupt shape of helix
Affects enzyme binding
Often show preference for minor or major groove
Intercalating can inhibit topoisomerases
What is proflavine and what does it treat?
Intercalating antibacterial drug - used in WW1 to treat deep face wounds
Completely ionised at physiological pH - ion-ion pair anchor drug in
What does doxorubicin treat?
Anticancer drug
Where does doxorubicin bind?
At the major groove in DNA, charged amino group acts as anchor
Why is doxorubicin considered a Topoisomerase ll poison?
Because intercalation prevents normal action of enzyme
Crucial to replication and mitosis
Briefly describe the process of gene expression
Cell transcribes the nucleotide sequence of a gene into an RNA molecule.
Cell then transcribes RNA molecule into amino acid sequence of a protein.
Define what a gene is
A gene is a segment of DNA that contains the instructions for making a particular RNA and/or protein.
What is an organisms karyotype?
The full set of chromosomes
Karyotype of humans is 46 chromosomes
What is interphase?
Cell is actively expressing its genes, DNA can replicate and complete gene expression to proteins.
What is M-Phase?
The mitotic phase where chromosomes become more densely packed - cell division
What’s significant about the structure at the telomere?
Higher concentration of G-C base pairs - chemical knot tying the ends
What’s the purpose of the centromere?
Attaches duplicated chromosomes to mitotic spindle
Why are genes found far from the centromere?
To reduce error
What’s the complex of nuclear DNA with histone and non-histone proteins called?
Chromatin
What is a nucleosome?
Nucleosomes contain DNA wrapped around a protein core of 8 histone molecules - 2 molecules each of histones H2A, H2B, H3 and H4
How many base pairs are in a histone octamer?
147 base pairs - 1.7 turns of DNA in a left-handed coil per histone octamer
What are the properties of histones?
Long N-terminal tail
High proportion of +ve amino acids - Arg and Lys
- Positive charge helps histones bind tightly to -vely charged sugar-phosphate backbone
What’s the role of topoisomerase l?
Relieve torsional stress of supercoiled DNA - only cleaves 1 strand
What’s the role of topoisomerase ll?
Cuts both DNA strands which are pulled apart to allow second strand to pass through - strands are then resealed
By what mechanism does topoisomerase ll break the sugar-phosphate backbone?
How do intercalating drugs interfere with topoisomerases?
By preventing replication transcription [artly by inhibiting topoisomerases function
What does camptothecin treat?
Cancer - topoisomerase poison
What’s the method of action for camptothecin?
Stabilises the cleavable complex formed between DNA and topoisomerase l
Cancer cell dies
What are fluoroquinolones?
Topoisomerase poisons
They’re synthetic agents that target topoisomerase lV, bacterial homologues
What is the method of action of fluoroquinolones?
They inhibit replication and transcription of bacterial DNA by stabilising the bacterial topoisomerase - DNA complex
Binding site only appears after the DNA has been ‘nicked’ and DNA strands are ready to be crossed over
How do fluoroquinolones lock to the enzyme?
Have a carboxylic acid which becomes charged at physiological pH as well as 2 R groups on opposite side
Why are chemical modifications to histone tail important?
Because modifications control which genes are switched on/off
What does a methyl group at K 9 mean on a H3 histone tail?
Heterochromatin formation, gene silencing
What does a methyl at K 4 and acyl at K 9 mean?
Gene expression
What does a phosphate at S 10 and acyl at K 14 mean?
Gene expression
Define epigenetic inheritance
The transmission of a heritable pattern of gene expression from one cell to its daughter cells that does not involve the DNA sequence
How can epigenetic transmission occur?
Parental cell divides, leaving half of the modified nucleosomes for each daughter cell.
Proteins that recognise the same modifications they catalyse can then restore the parental modification pattern of nucleosomes - helping eukaryotic cells to ‘remember’ whether the gene was active
How many bases are wrong in the entire process of cell division?
1 - 3 letters wrong
What is structural important at replication origins?
Stretches of DNA rich with A - T
How many replication sites are in the human genome?
10,000 replication origins
What protein opens the DNA helix?
DNA helicase
What way can DNA only be synthesised in?
5’ —> 3’ direction
What direction is DNA always read in?
ALWAYS reads 3’ —> 5’ direction
What has DNA Polymerase evolved to do?
Evolved to use nucleotide triphosphates as substrates which are paired to the base in the template strand to grow the chain in 5’ —> 3’ direction
How is proofreading achieved?
Polymerisation and proofreading steps are tightly coordinated - happening in different domains
If incorrect base is paired then it is removed and correct nucleotide is added and synthesis continues
What’s the proofreading domain known as?
A nuclease domain - E
Results in error in 1 in every 10 million base pairs.
What is the strand that can grow in 5’—>3’ direction called?
The leading strand
What is the strand that can grow in 3’—>5’ direction called?
The lagging strand
Grown discontinuously using back stitching - forms Okazaki fragments
What’s the function of DNA primase?
Creates an RNA primer - short sequence which provided a base-paired 3’ end as a starting point for DNA polymerase.
What’s the function of DNA polymerase?
Helps grow the chain from the primer
Will form Okazaki fragments if on lagging strand template
What’s the function of nuclease enzymes?
Once the new Okazaki fragment reaches the primer of a previous fragment, nuclease cleaves off the primer
What’s the function of repair polymerase?
Replaces primer section with DNA
What’s the function of DNA ligase?
Joins sections of DNA together
What function group do all topoisomerase poisons possess?
A lactone ring - cyclic ester
What are chain terminating drugs?
Drugs that act as false substrates and are incorporated into the DNA chain during replication
Chain can no longer be extended once added
What must chain terminating drugs have?
A triphosphate group
A structure that makes it impossible for further building blocks to be added - No 3’ OH group to build from
Also must be recognised by DNA template and interact with nucleic acids.
What drug is used to treat herpes? How does it achieve this?
Acyclovir - antiviral agent
Is a substrate for viral thymidine kinase but not human - converting it to its monophosphate.
Why can the triphosphate not be used in chain terminating drugs?
Cannot be used because drug would be too polar to enter cells
After the mismatch repair mechanism what’s the overall efficiency of DNA replication?
1 error in every billion base pairs
How does sickle-cell anemia occur?
Occurs as a result of one uncorrected error in the gene for Beta-globin
What’s the significance of thymine dimers?
Ultraviolet radiation causes this
Changes the structure of DNA - meaning those who cannot correct this have to avoid sunlight and are very susceptible to skin cancers
What two reactions are the most common to create serious DNA damage in cells?
Depurination - loses purine as polymerase skips over it - frameshift
Deamination - cytosine amine is replaced by ketone - forming T not G as product
How does the mismatch repair system recognise a mistake?
Because a mutation can alter the secondary structure of DNA
Hence repair proteins can recognise this
What’s the mechanism for the mismatch repair system?
1 - nuclease protein specific to the damage cleaves the phosphodiester linkages of the damaged nucleotide
2 - repair DNA polymerase replaces the gap created with correct nucleotide
3 - ligase enzyme joins the sections together
What do alkylating and metallating agents do?
They often form covalent bonds to nucleophilic centres in DNA with their highly electrophilic groups.
This can disrupt/prevent replication and transcription - useful against cancer
What are possible toxic side effects of alkylating and metallating agents?
Protein alkylation
How are inter-strand crosslinks formed?
React with nucleic acid base on each chain of DNA duplex
This crosslinks the strands - disrupting replication and transcription
How are intracellular-strand crosslinks formed?
Link two nucleophilic groups on the same chain
That DNA portion then becomes masked from enzymes - stopping replication
What does a drug need to form inter/intra-strand crosslinks in DNA duplexes?
TWO alkylating groups
How can you find basic nitrogen’s on nucleic acids?
Nitrogen’s that have lone pairs orthogonal to the ring - not involved in aromaticity
How does miscoding lead to altering protein structure and function? Where can this be useful?
Guanine exists in keto form - though when alkylated it exists in enol form
This makes it more likely to bind to thymine - useful in anticancer and antiparasitic drugs
What anticancer drug contains a aziridinium ion?
Nitrogen mustards, Chlormethine - crosslinks DNA so stop replication
(Aziridinium ion HIGHLY electrophilic)
What do nitrosoureas treat and how do they function?
Anticancer drug
Nitrosoureas decompose spontaneously in the body to form 2 active compounds
Give an example of a nitrosourea?
Carmustine
Lomustine
Streptozotocin
What do the active compounds of nitrosoureas do?
One carbamoylates lysine residues - inactivating DNA repair enzymes
Other causes alkylation on O6 of guanine and N3 of cytosine
What is Busulfan and what does is do?
Anticancer drug
Causes inter-strand crosslinking between guanine units.
What’s the general structure of Busulfan? What’s its role?
Busulfan has 2 sulfonate groups at either end of a 4 carbon chain
Sulfonate groups are good leaving groups and so can crosslink 2 DNA strands, stopping replication
(Inter-strand crosslinking)
What anticancer drug is neutral and unreactive?
Cisplatin
How is cisplatin activated and what’s its function?
Activated within cells by aquation
Intra-strand crosslinking — N-7 and O-6 positions of adjacent guanine molecules
What is Calicheamicin Y1 and what does it do?
Anticancer agent that cuts DNA strands and prevents DNA ligase from repairing the damage
How does Calicheamicin Y1 act as a chain cutter?
It creates radicals on DNA structure that react with oxygen to form peroxy species and DNA chain fragments
Where does Calicheamicin Y1 bind?
Binds at the minor groove in DNA helix
What compounds work the same way as Calicheamicin Y1?
Bleomycins and podophyllotoxins
