TBL 2 DNA Flashcards
DNA stored in the _______ is always ______ before the cell undergoes mitosis.
stored in the nucleus; replicated
_________ replication suggests that the parental DNA strands separate and serve as a template strand for replication, and do not re-anneal with each other. All daughter molecules contain ___________.
Semi-conservative replication;
all daughter molecules contain 1 parental strand and 1 daughter newly-synthesised strand
DNA replication always proceeds in the __’ to __; direction.
5’ to 3’
parental strand is read in 3’ to 5’ direction
DNA replication requires a ______ because DNA Polymerase cannot initiate DNA synthesis on their own, they can only add deoxyribonucleotides to the 3’ end of an existing chain.
Primers (usually RNA)
There are 5 classes of DNA polymerases (a, B, y, d, E). DNA polymerase __ is the main DNA polymerase for DNA replication.
DNA Polymerase delta
The bond between 2 nucleotides is a _________ bond, catalysed by DNA polymerase.
phosphodiester bond (between the 5’ phosphate group of the incoming dNTP and the 3’ OH group of the elongating strand)
Hydrolysis of ________ releases pyrophosphate and a large amount of energy needed to drive DNA replication reaction.
Hydrolysis of triphosphate on the incoming dNTP
Dideoxycytidine/zalcitabine and Azidothymidine/zidovudine are nucleoside analogues, which are used as inhibitors to _________ and therefore a HIV antiviral.
These analogues do not provide a free 3’ OH group for DNA P to extend and thus terminates chain replication.
Reverse Transcriptase
_________ are used as a drugs which has similar structure to DNA nucleosides, and therefore can be added to stop DNA replication.
Nucleoside analogues
Acyclovir (nucleoside analogue) is an antiviral drug used for ________, ______.
herpes simplex infections, chicken pox and shingles
Cytarabine/cytosine arabinose (nucleoside analogue) is a chemotherapy medication for various ________.
leukaemias
DNA replication begins at one or more specific _____ sites on the DNA molecule known as _________.
A-T rich sites; origins of replication
In prokaryotes, how many oriC are there?
single unique origin
replication proceeds bidirectionally from oriC simultaneously
In _________, there are multiple origins of replication for the long, linear chromosome.
eukaryotes
______ are a family of proteins which bind to one strand of DNA and use ____ as an energy source to break the hydrogen bonds holding the two strands together.
Helicases; ATP
____________ temporarily stabilises the unwound single-stranded portion of the DNA molecule.
This is cooperative binding, and is to prevent the ssDNA from reannealing to each other.
It also prevents ssDNA from being _______ by exonucleases.
Single-strand binding proteins (SSB proteins); degraded
_________ are proteins which cleave a strand of the helix to create a transient single-stranded nick, and allow free rotation around the intact strand to relieve strain on the DNA molecule, then resealing the broken strand.
Topoisomerases
_______ (specialised RNA polymerase) will synthesise a short RNA primer using the template strand to provide a free 3’ OH end which DNA P can extend.
DNA Primase
The RNA primer is transient, and will later be removed by _______ with _____ exonuclease activity and replace with a DNA version.
DNA Polymerase; with 5’ to 3’ exonuclease activity
As the templates for the two new daughter strands are _________, the replication fork is asymmetrical. Both daughter strands are only synthesised in the 5’ to 3’ direction, so there are two types of strands - _______ and _______.
antiparallel; leading and lagging
The ______ strand is synthesised continuously as a single polymer towards the replication fork; whereas the _______ strand is synthesised discontinuously in a series of _________, each synthesised (towards/against) the direction of the replication fork.
Leading strand;
Lagging strand; Okazaki fragments; each synthesised AGAINST the overall direction of the replication fork
______ catalyses the formation of phosphodiester bonds between the 3’ end of the new Okazaki fragment (replaced with dNTPs) and the 5’ end of the growing DNA strand with ATP hydrolysis.
DNA ligase
Which of the following requires ATP?
- DNA ligase
- DNA Polymerase
- DNA primase
Answer: ALL
_______ of the template for the (leading/lagging strand) allows both daughter strands to be synthesised in a coordinated manner.
Looping of the template for the LAGGING strand
=> allows both the DNA P to be in the same orientation
DNA P has a _______ mechanism to ensure no mistakes are made in the process.
proofreading mechanism
Another DNA repair mechanism is _______ repair, where deformities in the double helical structure due to incorrect base-pairing are recognised after replication is complete.
Enzymes determine the template and non-template strand and excise the wrongly incorporated base then replacing it with the correct nucleotide.
mismatch repair
All cells in the human body contains the same genetic material (all the genes), but serve different functions due to differences in ___________.
gene expression
The complete DNA sequence of an organism is known as its ______.
genome
The human genome is arranged into ___ chromosomes. __ autosomal pairs, 1 pair of sex chromosomes.
46 chromosomes; 22 autosomal pairs and 1 pair of sex chromosomes
_______ genes are genes which are being constitutively expressed by all cells, as they are needed for normal cell function.
Housekeeping genes
DNA replication and transcription occurs in the ______; whereas translation occurs in the _______.
DNA replication and transcription: nucleus
Translation: cytoplasm
The template DNA strand in transcription is known as the _______ strand. It is read from the _______ direction and is complementary to the mRNA produced.
antisense strand; read in the 3’ to 5’ direction
The non-template strand in transcription is known as the ______ strand, and it has the same sequence as mRNA, except that the T has been replaced by U.
sense strand
The promoter contains the _______ (5’-TATAAA-3’ on the non-template strand) and ___________.
TATA box and transcription start site
The TATA box is a binding site for _____ (general transcription factor) which facilitates the binding of RNA P.
TFIID
Is the promoter transcribed?
No, except for the transcription start site.
The terminator sequence is found at the end of the gene, coding for a polyadenylation signal sequence: 5’ _______ 3’ in the pre-mRNA.
5’-AAUAAA-3’
Transcription factor binding sites lie (upstream/downstream) of the promoter sequence and control the rate of transcription via transcription factor binding.
upstream
There are 3 types of RNA P.
RNA P 1: Transcribes ____ genes
RNA P 2**: Transcribes genes encoding proteins into ____
RNA P 3:
Transcribes _____ and 5S RNA genes
RNA P 1: Transcribes rRNA genes
RNA P 2**: Transcribes genes encoding proteins into mRNA
RNA P 3:
Transcribes tRNA and 5S RNA genes
_________ are required for RNA P to bind to its promoter and initiate transcription, achieving a basal rate.
General/basal transcription factors
Transcription factors can assess the DNA base pairs of the promoter and TF binding sites via the _______ and _______ of DNA, but the main point is through the _______.
major and minor grooves; mainly through major grooves
TFIID contains the _________ and TBP accessory factors (TAFs), and upon binding to the _______, it partially unwinds the DNA helix and widens the minor groove to allow extensive contact with DNA.
TATA binding protein (TBP); binds to the TATA box
After TFIID binds to TATA box, TFII__ and TFII__ binds, followed by the binding of TFII__, TFII__, TFII__ and TFII__.
1) TFIID
2) TFIIA and TFIIB
3) TFIIE, TFIIF, TFIIH and TFIIJ
Binding of TFII__ promotes further unwinding of the DNA helix to facilitate RNA synthesis, with kinase activity phosphorylating _____ residues on RNA P II to activate it for transcription.
TFIIH
- promotes further unwinding of helix
- activates RNA P by phosphorylating serine residues
RNA P _____ the unwound DNA helix behind it, dissociating the growing RNA chain from the template.
reanneals
The transcribed terminator encodes a polyadenylation signal sequence in the RNA, and transcription continues until ___ to ___ bp downstream of the signal sequence.
Proteins bind to cut and free the pre-mRNA from RNA P II.
10 to 35 bp
Histone acetylation will (reduce/increase) rate of transcription.
increase rate of transcription
=> Acetyl groups are negatively-charged. Acetylation will neutralise positive charge of histones, causing histones to bind less tightly to DNA => more accessible for transcription factors,
Histone deacetylation will (reduce/increase) rate of transcription.
reduce rate of transcription
Protein ____ is an inhibitor of transcription factor NFKB, which upregulates cytokine genes in the nucleus to promote inflammation.
How does aspirin, an anti-inflammatory drug, work?
IKB
Aspirin will inhibit the breakdown of IKB, which will bind to the TF NFKB, such that it remains in the cytoplasm and is unable to initiate the transcription of the cytokine genes, minimising inflammation.
Oestrogen receptors are ________ which regulate oestrogen regulated genes.
Over 70% of breast cancers overexpress the oestrogen receptor, resulting in uncontrollable tumor growth and proliferation.
transcription factors
Anti-oestrogen drugs like _______ act as progenitors of highly competitive inhibitors of the oestrogen receptor to repress its activity and prevent overactivation of genes controlling cell cycle progression.
Tamoxifen
Prokaryotes only have one type of RNA P.
The transcription factor (known as ____ factor) binds to the ________ to initiate transcription.
sigma factor binds to the Pribnow box to initiate transcription
RNA processing occurs in the _____.
nucleus
_____ are protein-coding sequences which form part of the mature mRNA; whereas _______ are nucleotides inserted between exons which are non-coding and thus edited out of the mature mRNA.
Exons; introns
5’ end of pre-mRNA is modified by the addition of a cap structure consisting of ___________.
a methylated guanine nucleotide/methylguanosine triphosphate
How is the 5’ methylated guanosine cap added?
It is formed by the hydrolysis of terminal triphosphate of mRNA to a diphosphate, which then reacts with the 5’ phosphate of GTP to form a 5’-5’ phosphate linkage.
This is further modified by methylation at the N7 position in the purine ring to form a 7-methylguanylate cap.
3 functions of 5’ cap:
- Protects mRNA from _______ by hydrolytic enzymes like nucleases.
- Defines the 5’ end of the mRNA, which serves to recruit the _______ for translation initiation.
- Distinguishes mRNA from other types of RNA.
- degradation
2. binding to the 40S ribosomal subunit for translation initiation
Viruses like ________ can interfere with the recognition of the cap during translation, thus blocking the formation of the translation initiation complex and preventing vital protein synthesis.
polioviruses
After the pre-mRNA has been cleaved at a site 11-30 bp downstream of the polyadenylation signal sequence, most 3’ ends are modified by addition of a series of about 200 ______ nucleotides catalysed by the ___________.
adenine nucleotides, catalysed by poly(A)-polymerase
Function of the 3’ polyA tail:
- Protects mRNA from _______ by nucleases
- Required to facilitate export of mRNA out of nucleus via _______.
- degradation
2. nuclear pores
Introns start with the splice donor site containing the sequence 5’___3’ and end with a splice acceptor site containing the sequence 5’___3’.
Splice donor site: 5’-GU-3’
Splice acceptor site: 5’-AG-3’
__________ are large complexes comprising of several subunits called snRNPS which contain small nuclear RNAs and sets of proteins.
Spliceosomes
RNA Splicing:
First protein is ___, which binds to the splice donor site of intron.
After which, the binding of ___, ___, ___ and ___ complete the formation of the spliceosome, which cleaves the splice donor sequence at the 5’ end.
- U1 to splice donor site (5’ GU 3’)
- U2, U4, U5 and U6 bind
=> spliceosome - Cleaves splice donor sequence
An ______ residue in the intron is used as a branch point in the intermediate step, binding to the __’ end of the intron.
(2’ OH group) adenine residue within the intron; binding to the 5’ end of the intron (G)
Phosphodiester bond between G at the end of the intron (i.e. the splice acceptor site) and the next exon is cleaved, and the intron is removed as a ______-like structure.
lariat-like structure
After the introns are removed, simultaneous ______ of the exposed adjacent exon sequences occurs.
simultaneous ligation
________ is a group of inherited autosomal recessive disorders in which there is an imbalance in the relative amount of globin chains.
Thalassaemia
In B-thalassaemia, there is a relative deficiency of ___-globin chains.
Deficiency of B-globin chains
Several types of B-thalassaemia is due to ________ mutations in the B-globin genes, resulting in improper mRNA splicing and thus production of non-functional B-globin chains.
splice site mutations
Patient with B-thalassaemia presents with severe anemia and hepatosplenomegaly. There is iron overload (__________) due to chronic anaemia, causing hepatic fibrosis and cirrhosis, darkening of skin.
haemosiderosis
The genetic code is made of ___ codons, which code for ___ amino acids.
64 codons; 20 amino acids
The genetic code is _________, meaning a single amino acid may be coded by more than one different codon.
degenerate
Third base position in a codon is a _____ position as base pairing at the 3rd base is not so specific.
wobble position
The start codon is ____, which codes for amino acid _______.
AUG - methionine
The stop codons are: ____, ____, ____. They do not code for any amino acids.
UAA, UGA, UAG
The mRNA template is read by the ribosome in the ____ direction.
5’ to 3’ direction
i.e. the ribosome binds to the 5’ end of the mRNA and looks for the start codon AUG
tRNA molecules have an anticodon loop that will bind to specific mRNA codon via ___________.
complementary base pairing
The 3’ end of the tRNA (____) is the ________ attachment site that binds to an amino acid via an _____ bond.
3’ end - CCA stem;
amino acid attachment site; via an ester bond
The tertiary structure of the tRNA refers to the actual twisting and folding of the secondary structure into a compact 3D ______ structure maintained by hydrogen bonds.
L-shaped structure
The 3D structure of tRNA is recognised by the enzyme ____________, which catalyses the formation of an ester linkage between the CCA stem and specific amino acid’s carboxyl group –> forms _________.
enzyme amino-acyl tRNA synthetase; forming aminoacyl tRNA
The binding interactions of the tRNA with the third mRNA codon base is weaker (wobble) and tolerates other bases in that position.
-
The active site of amino acyl-tRNA synthetase is complementary to the 3D conformation of the _________ and the __________.
1) specific amino acid
2) specific anticodon of the tRNA
Ribosomes are large ribonucleoprotein complexes composed of ________ and _______.
ribosomal proteins and ribosomal RNA (rRNA)
Each eukaryotic ribosome consists of two subunits:
Large (60S) and small (40S) ribosomal subunits
The large ribosomal subunit contains 3 binding sites for tRNA.
1) A site (holds the incoming tRNA carrying the next AA to be added)
2) P site (holds the tRNA carrying the growing polypeptide chain)
3) E site
The small ribosomal subunit contains the binding site for _______.
mRNA (5’ end)
rRNA in the large ribosomal subunit has _________ (enzyme) activity and catalyses the formation of peptide bonds between the amino acids.
peptidyl transferase activity
Pre-initiation complex for translation involves ________ binding to the (small/large) ribosomal subunit, positioning the tRNA-met to the __ site.
eukaryotic initiation factors (eIF-2) binding to the small subunit of the ribosome, positioning tRNA-met to the P site
eIF-__ and eIF-__ bind to the 5’ cap of mRNA, and are recognised by the pre-initiation complex, leading to the 40S subunit of ribosome binding to the mRNA.
eIF-4E and eIF-4G
Binding of the 60S large ribosomal unit occurs with the dissociation of ____ with the hydrolysis of ____, leading to conformational changes.
dissociation of eIFs with the hydrolysis of GTP to GDP –> conformational change occurs
The translocation of tRNA occurs along the ribosome, guided by the elongation factors with the hydrolysis of ___ to promote the movement of ribosome.
hydrolysis of GTP promotes movement of ribosome along the mRNA
Termination of translation: When the in frame stop codons (___, ___ and ___) reach the A site of the ribosome, a protein ________ binds directly to the stop codon at the A site. (There are no tRNAs with anticodons complementary to the stop codons.)
UAA, UAG and UGA
Protein release factor
Bound ribosomes on the rough ER are responsible for synthesising ________ and ______ proteins.
secretory and transmembrane proteins
Polypeptides of proteins destined for secretion (i.e. the endomembrane system) are marked by a __________ (first 20-24 amino acids rich in hydrophobic amino acids), targeting them to the ER.
signal sequence/peptide
Marking polypeptides for secretion:
The signal peptide near the N-terminus of the polypeptide is recognised as it emerges from the ribosome by a ____________ (protein-RNA complex).
Signal recognition particle (SRP)
The ___________ is an adaptor which brings the ribosome to the rER membrane by binding to it, causing translation to resume. The growing polypeptide chain is synthesised into the lumen of the rER.
Signal recognition particle (SRP)
(Secreted/transmembrane) proteins are translocated fully into the rER lumen during translation.
These proteins are completely contained within the vesicle and directed towards the GA or other organelles.
Secreted
(Secreted/transmembrane) proteins have an additional hydrophobic sequence which holds them in the ER membrane.
These proteins are anchored to the vesicle membrane such that when the vesicle fuses with another membrane, the protein is embedded within the membrane.
Transmembrane
Once the secretory and transmembrane proteins are translated, the enzyme _________ cleaves the signal sequence on the N-terminus of the polypeptide, and the protein folds into its native conformation.
signal peptidase
__________ (antibiotic) inhibits initiation of translation by binding to the _________ of the prokaryotic ribosome. This interferes with the binding of fMet-tRNA to the 30S subunit during initiation.
Streptomycin; binds to the 16S rRNA of the 30S small ribosomal subunit
_________ inhibits aminoacyl-tRNA binding by binding to the 30S ribosomal subunit.
Tetracycline
________ inhibits translocation of the ribosomal complex by binding to the 50S ribosomal subunit.`
Erythromycin (E for Eukaryotic factors which help with translocation)
Chloramphenicol inhibits ___________ in the 50S large ribosomal subunit, preventing polypeptide elongation.
Peptidyl transferase
_________ terminates elongation prematurely, acting as an analogue of the 3’ end of aminoacyl-tRNA.
Puromycin (P for premature termination)
In glycosylation of polypeptides, pre-assembled carbohydrate chains are N-linked to asparagine (Asn) of the _________ sequence by (enzyme).
This stage occurs in the ______ (organelle), as glycan on the lipid-linked precursor exist within the ____ lumen.
Asn-X-Ser/Thr sequence (where X is any AA except for proline)
By glycosyl transferases
Occurs in the rER; rER lumen
Glycan addition occurs in the ________, whereas glycan processing occurs at the ________. (where carbohydrate chains are processed by trimming followed by extension)
ER; golgi apparatus
Prenylation/acylation of polypeptides refer to addition of _____ groups.
lipid groups
Insulin (protein hormone) biosynthesis:
- _______ bond formation from the oxidation of two cysteine residue side chains. (preproinsulin —> proinsulin)
- Proteolytic cleavage (i.e. removing a sequence of amino acids):
Proinsulin is cut _____ after the disulfide bonds are formed, and ________ is removed from the middle of the protein in a secretory vesicle.
=>Active insulin consisting of two polypeptide chains.
- Disulphide bond formation
2. Proteolytic cleavage - Proinsulin is cut twice and the connecting peptide C chain is removed.
________ marks intracellular proteins for proteolysis (degradation) by the proteasome.
Ubiquitin
Endocrine: __________ produced in the hypothalamus acts on the kidneys.
Vasopressin
Endocrine:
_______ produced by the adrenal glands act on the trachea.
Adrenaline
_______ effect is when the hormone acts on an adjacent cell.
Paracrine
Paracrine:
Insulin produced by the B-cells in the islets of Langerhans acting on _________ to inhibit glucagon secretion.
neighbouring alpha cells
Paracrine:
________ and ______ produced by the endothelial cells in blood vessels, causing vasoconstriction.
Endothelin-1 and nitric oxide
Autocrine:
Activated T-cells secrete _____, which binds to the receptor on the same cell, signalling the cell to enter the cell cycle and inducing cell proliferation and differentiation.
IL-2
Cells can communicate via signalling between __________ via physical contact.
E.g. TCR on T-cells binding to MHC class II complexes on APC.
membrane-attached proteins (MAPs)
Intracellular receptors will receive signals from _________ ligands.
hydrophobic ligands
that can cross the membrane
________ receptors are membrane receptors which allow specific ions to pass through when the receptor changes its conformation due to a ligand binding.
Ionotropic receptors (ion channel receptors)
Acetylcholine is a ligand to the sodium ion channel receptor __________ on skeletal muscles.
Nicotinic acetylcholine
_______ is a general anaethesia that is administered to compete with acetylcholine for binding sites on the nicotinic acetylcholine receptors, leading to reduced muscle contraction.
Atracurium
Structure of GPCRs: 7 transmembrane _______
7 transmembrane a-helices
G-protein has __ subunits.
Active G-protein will dissociate into two active components:
3 subunits
2 active components: a and By subunits
Internal GTPase activity of the __ subunit of G protein will cause the hydrolysis of GTP back to GDP.
alpha subunit
___ protein-linked receptor: B1 adrenergic receptor
Ligand:
Pharmocology:
Gs protein-linked receptor
Ligand: Adrenaline, noradrenaline
Pharmocology: Beta blockers
G_ protein activates adenylyl cyclase.
G_ protein inhibits adenylyl cyclase.
Gs protein
Gi protein
________ catalyses the synthesis of cAMP (second messenger).
cAMP then activates ________, which phosphorylates other proteins and may lead to different effects depending on the linked G protein.
Gi/Gs protein –> Adenylyl cyclase —> cAMP —> Protein Kinase A (PKA) –> phosphorylates other proteins
Gq proteins activate _______, which cleaves PIP2 into ___ and IP3.
IP3 binds to IP3-gated _______ channel in the ER membrane, causing ___ ions to diffuse out of the ER and into the cytoplasm.
Gq proteins –> Phospholipase C (PLC) –> cleaves PIP2 into DAG and IP3 (second messengers)
IP3 binds to gated Ca2+ channels, causing Ca2+ ions to diffuse out of the ER into the cytoplasm and activate calcium-sensitive proteins to produce a cellular response.
Dopamine is a ligand that binds to ___________ receptor, which is G_ protein linked.
Receptor: D2 dopaminergic GPCR
Gi protein linked
Glucocorticoid receptor is an _________ receptor. Its ligand is ______.
Glucocorticoid receptor - intracellular
Ligand: Cortisol, corticosterone
Thyroid hormone receptor is an _________ receptor. Its ligands are:
Thyroid hormone receptor - intracellular
Ligands: T4 (thyroxine) and T3 (triiodothyronine)
Carcinogens can cause DNA damage in the following ways:
1.
- (Physically link two DNA bases)
- (Chemical/oxidate changes around the bases leading to non-recognition of DNA)
- (Covalently link two DNA molecules via G/A)
- Double and single strand breaks
- Base dimer
- Base hydroxylations and abasic sites
- DNA adducts and alkylation
___________ are common environmental pollutants formed from the combustion of fossil fuels or tobacco with complex, aromatic multi-benzene ring structure.
Polycyclic Aromatic Hydrocarbons (PAH)
E.g. Benzo[a]pyrene (B[a]p)
PAH:
Benzo[a]pyrene (B[a]p) undergoes a two-step ________ process which activates it.
Enzyme 1: ________ oxidises B[a]p to an epoxide, a strained molecule which is highly reactive.
Enzyme 2:
________ splits the functional group of the epoxide into two hydroxyl groups. [detoxification pathway protective mechanism]
The resulting dihydrodiol becomes a substrate for _____, which oxidises it to an epoxide with a dihydrodiol –> extremely reactive and electrophilic.
Epoxidation;
Enzyme 1: P450 enzyme;
Enzyme 2:
Epoxide hydrolase
Resulting dihydrodiol becomes a substrate for P450, forming an extremely reactive epoxide.
DNA is nucleopholic/electrophilic.
nucleophilic (due to the large source of electrons)
PAH:
__________ is a potent human liver carcinogen.
It is a substrate of P450, which oxidises it to form an epoxide.
This will cause it to be highly reactive/electrophilic, reacting with DNA bases (particularly ______) to form DNA conjugates/adducts.
Aflatoxin-B1;
reacts with DNA bases to form guanine-alfatoxin B1 conjugate (adduct)
PAH:
___________ is a potent human bladder carcinogen.
It is a substrate for P450 (CYP1A2), which converts it to a _________. (DNA-damaging)
2-napthylamine; converted to N-hydroxy derivative (DNA-damaging)
Body’s mechanism of coping with mutagens:
Phase I: Addition of ___________ to generate an intermediate (may be DNA-damaging)
Phase II:
Conjugation of phase I functional groups; generating _________ metabolites that can be safely excreted from the body.
Phase I: Addition of functional groups to generate intermediate
Phase II:
Conjugation of functional groups which may generate polar, harmless metabolites
2-napthylamine Phase II enzyme system involves __________ adding sugar molecules to the amines, detoxifying the molecule –> non-toxic, polar and excretable in urine.
However, as urine pH is (acidic/alkaline), this breaks up the molecule (losing the sugar), generating a (positively/negatively)-charged nitrenium ion that is highly reactive in the urine stored in bladder.
=> attack bladder _______ cells, causing mutations and tumors.
2-napthylamine:
Phase II - Glucuronyl Transferase adding sugar molecules
urine pH acidic => breaks up molecule => positively-charged nitrenium ion => highly reactive in the urine => attack the bladder epithelial cells
Solar (UV) radiation results in the formation of __________ between the adjacent thymine bases in DNA.
formation of pyrimidine (thymine) dimers
=> no longer recognised as thymine molecules, leading to mutations
_________ radiation generates free radicals in cells.
Oxygen free radicals possess unpaired, single electrons and are highly (nucleophilic/electrophilic), targeting nucleophilic DNA molecules.
This may result in __________, base removal and base modifications.
Ionising radiation
Free radicals are highly electrophilic.
May result in double/single strand breaks, base removal and base modifications
____ protein deals with cellular stresses by initiating a series of events to fix the problem.
p53 protein
DNA repair
There are 5 types of DNA repair mechanisms:
- Direct reversal
- Base excision repair
- Nucleotide excision repair
- Mismatch repair
- Recombinational repair
Direct reversal DNA damage repair:
_______ splits cyclobutene pyrimidine dimers.
__________ and alkyltransferases remove alkyl groups from bases.
1) Photolyase
2) Methyltransferases
Base excision repair occurs mainly for ________ damage.
- DNA _______ and endonucleases excise the bases.
- __________ fills the gap.
- _____ completes the repair.
apurinic/apyrmidinic damage
- DNA glycosylases and endonucleases excise the bases.
- Repair polymerases (e.g. Pol b) fills the gap.
- DNA ligase completes repair.
For base excision repair, DNA glycosylase will only remove the affected __________.
Endonuclease then cleaves the backbone to allow for the addition of the new nucleotide, before reforming the backbone by DNA ligase.
nitrogenous base (not the entire nucleotide --> sugar-phosphate backbone left intact)
Nucleotide excision repair occurs mainly for _______.
bulky DNA adducts (i.e. two DNA molecules covalently link together via G or A)
Nucleotide excision repair:
_________ proteins assemble at the damage site, excising a stretch of nucleotides on either side of the damage.
Xeroderma pigmentosum (XP) proteins
=> Deficiency of XP proteins severely predisposes individuals to skin cancer.
________ repair looks for mismatched base pairs in DNA due to incorrect complementary base pairing.
Mismatch repair
_________ repair is repairing double-stranded DNA damage by recombination with a homologous intact duplex (usually sister chromosome)
Recombinational repair
Cell-based: In vivo/in vitro
Cell-free: In vivo/in vitro
Cell-based: In vivo
Cell-free: In vitro
In-vivo (cell-based) DNA cloning first involves the in vitro construction of _________.
(1) Cut target DNA and replicon with the same ________ enzyme, so that the two ends of the DNA are compatible.
(2) Mix and join DNA fragments with DNA ligase.
(3) Transform recombinant DNA molecules into host cells. (e.g. bacteria)
(4) Selective propagation on agar plate
recombinant DNA molecules/plasmids
(1) Cut using restriction endonucleases
(2) Mix using DNA ligase
Restriction sites are usually 4-8bp _______ sequences.
palindromic = same sequence on both strands when read from 5’ to 3’ direction.
Restriction endonucleases produce either _____ ends or ______ ends.
Blunt ends (straight cuts with no nucleotide overhang) or sticky ends (staggered cuts with nucleotide overhangs which make annealing of DNA fragments more efficient)
Transformation of recombinant DNA molecules into host bacterial cells is done through ________ or _________.
heat shock or electroporation
What is the function of selecting colonies after bacterial transformation?
Colonies that are ultimately selected for are the transformed bacterial cells with recombinant plasmids containing the target DNA fragment.
Selection of colonies
1) Positive selection: Selection for transformed bacteria which __________
2) Negative selection: Selection for transformed bacteria which _________.
1) Positive selection: Selection for bacteria which have taken up plasmid
2) Negative selection: Selection for bacteria which have taken up recombinant plasmid
Recombinant bacterial colonies (should/should not) survive on amp+ tet+ agar plates.
Should survive on amp+
Should not survive on tet+ (as the insertion of DNA fragment into a plasmid results in disruption of the tet gene sequence)
DNA is negatively-charged, so it moves towards the (cathode/anode) during gel electrophoresis when electrical force is applied.
Anode - Positive electrode
__________ is a method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA molecules combine to form double-stranded molecules.
Nucleic acid hybridisation
In nucleic acid hybridisation, target DNA is immobilised on a solid support - _________ or _____ membrane.
nylon or nitrocellulose membrane
__________ refers to the power to distinguish related sequences during hybridisation between the probe and target DNA for nucleic acid hybridisation.
Hybridisation stringency
Hybridisation strigency (increases/decreases) with increasing temperature, and (increases/decreases) with increasing Na+ concentration.
- Increases with increasing temperature
- Decreases with increasing Na+ concentration
(i. e. more stringent = high temperature, low salt concentration)
_____ is an in-vitro method to selectively amplify a specific region of target DNA.
Polymerase Chain Reaction (PCR)
[in vitro - cell free]
_________ is a collection of microscopic DNA spots representing single genes, robotically arrayed on a solid surface.
DNA microarray
