Gary Sharples Flashcards
What is the difference between :
Dna A and dna A
The one with the capital is a protein the one in lower case is a gene.
What was the Meselson-Stahl experiment? What did it conclude?
- Bacterial cells grown for several generations on a medium containing heavy N15 nitrogen medium (DNA therefore contained heavy N15 DNA).
- the cells were then transferred to a lighter N14 medium.
- at various time the DNA was extracted and dissolved in a solution of caesium chloride and spun in centrafuge.
- conch gradient is established.
- first generation contained mix of light and heavy.
- second generation contained both LL and LH .
How was the discovery of bidirectional replication carried out?
Using Radioisotopes, the density was the same either side of the origin of replication. (If unidirectional it would only occur one side).
- grow e.coli in media without radioactively lab led thymidine.
- grow in 3H thymidine of low specific activity.
Grow 3H thymidine of high specific activity.
Explain the process of bidirectional replication
When DNA is stored in double stranded circle covalent lay closed. Replication begins at the origin of replication.
Semi conservative replication proceeds in 2 directions.
- Per circle there are 2 replication forks, each one with leading and lagging strand.
What is the difference between leading and lagging strand synthesis?
Leading strand is synthesised continually in the 5’ to 3’ direction.
Lagging strand is synthesised discontinuously in short pieces known as Okazaki fragments. Still in the 5’ to 3’ direction.
What do all DNA polymerases add nucleotides to the 3’ end of a growing strand?
- a template: strand to copy
- a primer: another strand annealed to the template tot supply a 3’ OH group
- dNTPs: deoxynucleoside triphospahte.
How is DNA polymerase capable of correcting mistakes?
It has a 3’ to 5’ exon lease activity, this means it only makes mistake 1/5*10^7 bp
Explain the process of DNA polymerase exotic lease activity.
- Polymerase miss airs dC with dT.
- Polymerase repositions the miss paired 3’ terminus into the 3’ to 5’ exonuclease site.
- Exonuclease hydrolysed the miss paired dC
- The 3’ terminus repositions back to the polymerase site
- Polymerase in-cooperates the correct nucleotide
How is DNA replication primed?
By RNA, the RNA primer (oligonucleotides) are synthesised by DNA primase.
- DNA polymerase then extends the RNA primers.
In bacteria what synthesises the RNA primers?
How long are the oligonucleotides that are synthesised?
DNA primase DnaG.
10-12 base pairs long.
How is the primer removed?
The primer is removed by DNA polymerases 5’ to 3’ exonuclease activity (this is different from the 3’ to 5’ used to correct bases). It syntheses a new DNA strand as the RNA is removed.
How are nicks in the DNA backbone sealed?
DNA ligase seals the nicks in the backbone between Okazaki fragments by catalysing the formation of a phosphorites tear bond. E.coli DNA ligase utilises nicotine midge (NAD) as a cofactors in this reaction.
How are parent strands separated at the replication fork?
DnaB helically. A 5’ to 3’ helicase that unwinds the parental DNA as it moves.
How does DnaB helicases structure enable it to carry out its function?
It consists of 6 identical subunits that forms a ring.
Of the 6 identical ATP binding sites 2 opposing ATP bind ATP tightly. 2 are more likely to bind ADP and phosphate and 2 are empty.
- these may inter convert as ATP is hydrolysed- this creates a ripple effect that continually runs around the ring.
- because of these conformaitonal changes the loops in the centre of the ring for DNA binding cause ossolation up and down.
- it’s thought the ossolation gloop may pull DNA though the central hole thus unwinding the double stranded helix.
Why is DnaG primase bound to DnaB helicases?
It ensures that DnaG is in the correct position for RNA primer synthesis. The activities of both protein are stimulated by this interaction .
What polymerase is used as the replicative polymerase in E.coli?
DNA polymerase III
What is OriC?
A 245 bop sequence necessary for replication.
- 5 repeats of a 9bp sequence make up the R1-5 for key initiator protein DnaA
- 3 additional repeats are bound by DnaA only when complex with ATP.
What is DUE at OriC?
The DNA unwinding element is called DUE. It is AT rich and the site where DNA is opened for replication.
How is the initiation of DNA replication regulated?
Hda stimulates ATP hydrolysis by DnaA to aid dissemble after initiation.
Dam methylates adenine at GATC in e.Coli
How is DnaB loaded onto the fork?
DnaB is loaded onto the replication fork by DnaC, it opens the DnaB ring. ATP hydrolysis releases DnaC leaving DnaB bound to the ring.
What is SSB?
An accessory protein.
- It stops unwound parent strands from reanealing.
- protects DNA strand from degradation or damage.
What does DNA gyrase (topoisomerase II) do?
Alleviates supercooling by cutting 2 strands of a DNA molecule and passing another though the break before reasealsing the cut strands.
What do the 3 DNA polymerases do in E.coli?
DNA polymerase 1: removes RNA primer and replaces it with DNA
DNA polymerase 2: filling gaps following repair of DNA damage
DNA polymerase 3: chromosome replication
What is the purpose of the B sliding clamp on DNA polymerase III?
Homodimer which encircles DNA.
Imparts process it’s by ensuring that the core polymerase does not fall off during DNA synthesis.
How is the B clamp loaded on to the lagging strand at each Okazaki fragment?
The y (gamma) complex of DNA pol III. - binding of ATP allows opening of the clamp whilst ATP hydrolysis allows clamp release and ring closure.
In very brief terms explain how DNA replication occurs in E.Coli.
8 steps
- DnaA opens the duplex at OriC.
- DnaC loads DnaB helicases.
- DnaG synthesises RNA primer on the lagging strand.
- Alpha subunit synthesise DNA on leading and lagging strand.
- Tau subunit ensures dimerisation of core polymerase subunits.
- Y complex loads B clamp at each Okazaki fragment
- B clamp encircles DNA and binds complex to fork.
How long does it take E.coli to replicate?
Should take around 40 mins but it can divide at much faster rates of around 20-25 minutes. 900 nt per second.
Why does DNA homologously recombine?
Conserves genetic identity - aligns chromosomes at meiosis - repairs DNA breaks - restores stalled replication forks Generates genetic diversity - rearranges genes within a genome - exchanges homologous partners - incorporates foreign DNA - Contributes to acquisition of new traits
Define; Crossover/ splice/ recombinant
Recombination where flanking markers are exchanged
Define; non-crossover/ patch/ non-recombinant
Recombination where flanking markers are not exchanged
Define; gene conversion
A mismatched DNA sequence from one heteroduplex DNA strand is replaced with a sequence complementary to the other strand resulting in aberrant gamete ratios.
How does DNA recombination aid recovery of problems that arise during DNA replication?
Partner chromosomes are found and single strands exchanged to form DNA branched structures. They often offer a template to Copt any genetic information that might be lost.
What is the holiday model?
Homologous chromosomes are nicked at identical locations, the strand strands from one side of the nick invades the homologous chromosomes base pairing with complementary strands.
- invading strands are covalently linked to the original strands strands ar the nicked points. This forms a holiday junction. The holiday junction migrates away from the nick points in a process known as branch migration. As it does so DNA is swapped between chromosomes. This creates heteroduplex regions on both chromosomes where minor base sequence differences between homologous chromosomes results in a region of DNA with low percentage of mismatched base pairs.
How and what are the results of the 2 different cleavage of Holliday junctions?
- if crossed strands are cleaved by an endonuclease then after ligation within the chromosome there will be 2 non-recombinant chromosomes.
- if one rotates 180 degrees a process called isomer action it’s easier to visualise how uncrossed stands are broken, this process results in recombinant chromosomes with short heteroduplex regions.
What is the difference between hetero/homoduplex?
Homo = a DNA molecule composed of 2 chains with each derived from the same parent molecule Hetero = a DNA molecule composted of 2 chains etched derived from a different parent molecule.
Explain the process of homologous dependant double strand break (DSB) repair. (SDSA)
Duplex is broken with double strand break.
- first the DNA undergoes nuclease degradation forming DNA duplexes with 3’ ended single stranded tails.
- one of the single stranded tails invades the non-broken duplex at a region of homology. Forming a region of heteroduplex.
- the displaced pink strand which is of the same polarity of the invading grey strand forms a loop the D loop (the displacement loop)
- the 3’ end of the invading strand acts as a primer for DNA synthesis the complementary strand serves as a template.
- a small bubble is formed consisting of the template, a small region of Newley synthesised DNA and the displacement strand.
- this continues until the new DNA complimentary to the double stranded break in the grey stand is synthesised.
What are the pre-synaptic stages of recombination?
Events take pales that are needed for the initiation of of recombination; this stage includes the formation of gaps, single or double strand breaks and the exposure of single stranded regions.
What are the synopsis stages of recombination?
The homologous strands are paired and strand exchange occurs between them to produce joint-molecule intermediates.
What are the Post-synapsic stages of recombination?
The joint molecule intermediates are processed to mature recombinant products.
What is branch migration?
It moves the joint along the DNA resolution: involves strand cleavage at the joint to separate the linked DNA molecules.
What is RecBCD involved in?
Is involved in end processing.
What are RecB/D
Helicases that travel along each of the strands at a DNA break.
RecB moves in the 3’ to 5’ direction, whilst recD move 5’ to 3’ on the opposite strand. Thus they unwind together as bipolar helicases.
What does the nuclease domain of RecB do?
Cuts both DNA strands as it passes through the RecBCD complex.
What occurs at a chi site?
Cutting of 3’ strand ceases, but continues on the 5’ strand, RecA can load onto the exposed ssDNA overhang
Explain the repair of a DSB with relation to chi sites.
8Bp chi sites are NOT randomly positioned throughout the E.coli chromosome. They are over represented and most are orientated away from the Ori.
- this promotes recovery close to a break at a replication fork.
- recombination at such breaks allows replication to restart mediated by PriA.
What is the purpose and function of RecA protein in E.coli?
Binds to ssDNA.
- polymerises on the ssDNA to form a helical nucleoprotein filament that can be seen my elctromicroscopy.
- it is the RecA nucleoprotein that catalysts the homologous DNA pairing and strand exchange states of recombination.
How does strand exchange by RecA occur in vitro?
Reactions require ATP and MG2+
- RecA polymerises at the ssDNA gap and initiates pairing with the 332P-labels linear duplex.
- strand exchange can be followed by removing proteins and analysis on agarose gel.
- strand exchange generates a holiday junction intermediate, but continues to the end of the duplex to generate complete strand exchange products.
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What is the function of RecQ?
RecQ is a helicase that functions at the at the interface between DNA replication and recombination to repair damaged replication forks.
What is the structure of RecQ?
Has a highly conserved helicase domain that comprises 400 AA.
- the RQC domain in unique to the RecQ family and consists of a zinc binding domain and a winged helix involved in DNA recognition.
Why do we use model organisms?
Life is diverse so studying a few organisms is easier
It allows an understanding of how proteins function together to produce life.
Organisms tend to be selected for ease of manipulation and experimentation.
How can RecQ aid replication?
- can be impeded by DNA secondary structure including hairpins or G-graduplexes
- RecQ helicases act to disrupt these structures and allow smooth replies one progression.
- RecQ helicase unwinds DNA with 3’ to 5’ polarity
- It typically catalysts Holiday junction branch migration, fork regression and also will unwind D-loops, triple helicase, hairpins and G-guadruplex DNA.
How are topoisomerases used to relieve torsional stress arising from DNA supercoiling?
By transient lay breaking the DNA backbone in one (tyope 1) or both (type 2) strands then passing intact DNA through the opening before resealing the break.
How does holiday junction resolution occur by dual strand scisson?
Once the 4-stranded holiday junction has formed it can branch migrate over long distances as the two paired DNA molecules are homologous.
- resolution occurs by nicking an opposing pair of strands at the point of the crossover.
What is Lex A involved in? What is the response?
Lex A regulates the expression of multiple genes involved in DNA damage repair, including RecA RuvAB and uvrAB.
This is the sos response
