Dna R, R And R

Front 1

What are the building blocks of DNA?

Back 1

Dexoyribonucleotide triphosphates

Front 2

What bonds link together nucleotides in DNA?

Back 2

Phosphodiester bonds

Front 3

How is the formation of phosphodiester bonds kept energetically favourable?

Back 3

The 3' OH group attacks the alpha phosphate on the dNTP, this release a pyrophosphate group.

The pyrophosphate group is rapidly broken down by pyrophosphatases to 2 inorganic phosphates. This increases entropy of the reaction and thus keeps the reaction favourable with a Delta G of -30KJmol^-1

Front 4

What is the polarity of DNA?

Back 4

5' to 3'

Front 5

How does density gradient CsCl ultracentrifugation separate DNA of different MWs?

Back 5

Under high centrifugal force, the Cs+ and Cl- ions split apart. The Cs+ ions are forced from the centre of the tube to the side, they are also pushed upwards, thus creating a density gradient.

If DNA is present in solution it will migrate to its isopycnic point where it has the same density as the solution.

Front 6

Which dye is used to label DNA before Density Gradient CsCl ultracentrifugation?

Back 6

Ethidium bromide.

Front 7

At what speed and for how long is DNA spun in Density Gradient CsCl ultracentrifugation?

Back 7

100,000rpm for 10 hours

Front 8

Other than the correct semi-conservative replication, which two theories were suggested for how DNA might replicate?

Back 8

Conservative replication = Replication where the double strands are not separated and where they are instead replicated together.

Dispersive replication = This is where patches of single-stranded DNA are opened up and replicated and they are joined together by recombination to generate new strands.

Front 9

How did Kornberg determine DNA polymerase activity?

Back 9

He used a 1000-5000bp poly-dA template and added a 10-20bp oligo-dT primer.

Then add 3H/14C5me-dTTP and a buffer at pH7.

We add acid because the 3H/14C5me-dTTP is soluble in acid but the template and primer are not. So the soluble nucleotides have to have joined to the primer to not dissolve.

We then wait for replication to occur before collecting the precipitate on a glass slide and counting radioactivity.

Front 10

How did Okazaki's pulse-chase experiment help develop the lagging strand hypothesis?

Back 10

He added a small amount of 3H-CH3 thymidine to E.coli as the pulse, he then added excess normal thymidine as the chase.

When he used a short pulse, there were both small and long radioactive fragments. Whereas, when he used a long pulse there were only long radioactive fragments.

This was found to be the Okazaki fragments on the lagging strand which had the gaps between them filled, explaining that the fragments appeared short after a short pulse but long after a long pulse.

Front 11

What structure does the lagging strand assume in DNA so that it can be replicated in a 5' to 3' like the leading strand?

Back 11

It assumes a trombone-like structure.

Front 12

Which enzyme (in prokaryotes) produces Okazaki fragments along the lagging strand?

Back 12

DNA primase/DnaG

Front 13

Which polymerase (in prokaryotes) fills in the gaps between Okazaki fragments?

Back 13

DNA polymerase III

Front 14

Which polymerase removes the RNA primers on DNA and replaces them with DNA?

Back 14

DNA polymerase I

Front 15

How does DNA ligase seal nicks in DNA?

Back 15

It forms a covalent bond with a 5' phosphate using NAD+ or ATP as a co-factor.

It transfers AMP to the 5' phosphate.

The 3'OH then completes nucleophilic attack on the AMP-phosphate bond. This regenerates the phosphodiester backbone and causes AMP release.

Front 16

What are the functions of DNA polymerase I?

Back 16

It is involved in lagging strand replication and DNA repair.

It has both 5' to 3' and 3' to 5' exonuclease activity.

Front 17

How many molecules of DNA polymerase I are there per cell and what is their turnover rate?

Back 17

400 molecules

Each enzyme has a turnover of 600bp/min.

Front 18

What are the functions of DNA polymerase II?

Back 18

It is involved in DNA repair.

It only has 3' to 5' exonuclease activity.

Front 19

How many DNA polymerase II molecules are there per cell and what is their turnover rate?

Back 19

The number of molecules per cell is unknown.

They have a turnover rate of 30bp/min.

Front 20

What are the functions of DNA polymerase III?

Back 20

It is the main polymerase involved in de novo DNA synthesis.

It only has 3' to 5' exonuclease activity.

Front 21

How many molecules of DNA polymerase III are there per cell and what is their turnover rate?

Back 21

10-20 molecules per cell, each with a turnover of 9000bp/min.

Front 22

What are the effects of conditional mutations in DNA polymerase I?

Back 22

These cells are only viable during slow growth and they are vulnerable to DNA damaging agents.

This is to say that they are only lethal in certain conditions.

Front 23

Are conditional mutations in DNA polymerase II lethal?

Back 23

These mutations are not lethal.

Front 24

Are conditional mutations in DNA polymerase III lethal?

Back 24

Yes, they are lethal.

Front 25

Give the 2 types of mutation related to replication machinery

Back 25

Quick stop = Replication stops immediately as the mutation affects elongation machinery or supply of necessary precursors.

Slow stop = This is where replication can continue to the end of the current cycle but cannot restart due to mutations in the initiation machinery.

Front 26

Give 2 examples of chemical mutagens

Back 26

Ethyl methanesulfonate

Methylnitronitrosoguanidine

Front 27

How are mutations in DNA replication machinery determined?

Back 27

We separate different proteins from a wild type cell using ion exchange or gel filtration chromatography. Or we take plasmids containing the gene for different the different replication proteins.

We then insert each of these into a fraction from the mutant. And we add 3H/14C5me-dTTP so that we can measure radioactivity and determine which fraction has DNA replication activity.

We can then determine which protein was needed to be added for replication to occur, therefore we can see which gene was the mutant.

Front 28

Give 2 examples of proteins identified by the aforementioned method (for DNA replication protein mutations)

Back 28

dnaB

dnaG

Front 29

How was Cairns autoradiogram initially used to view the bacterial chromosome?

Back 29

E.coli were grown on a medium of 3H thymidine.

They were then gently lysed and the lysate was spread on an electron microscopy grid and exposed to X-ray film for 2 months.

Front 30

What did we learn from Cairns' first autoradiogram?

Back 30

We learned that E.coli's chromosome is circular and that it has a single origin of replication (we saw this from the theta structure)

Front 31

What did the altered Cairns autoradiogram with low 3H thymidine then high 3H thymidine show?

Back 31

By growing E.coli on low 3H thymidine and then pulsing them with high 3H thymidine we get an X-ray film with lightly and heavily-labelled regions.

From this we determined that replication is bidirectional as there were heavily labelled regions on both sides of the replication fork.

Front 32

In Cairns' autoradiogram, what was the process used to gently lyse cells?

Back 32

A freeze-thaw technique is used but the E.coli are grown on a medium with plenty of KCl and glucose in order to reduce stress to the cells.

E Ohtsubo // Journal of Molecular Biology

Front 33

What are the 4 domains found in dnaA?

Back 33

A helicase loading dimerisation domain

A linker domain

An AAA+ domain (ATP binding, ATP hydrolysis, ssDNA binding and filament formation)

A dsDNA binding domain

Front 34

What is the process of dnaA filament formation at the origin of replication?

Back 34

dnaA binds sites called dnaA boxes at OriC. It initially binds high affinity dnaA boxes called anchoring sites.

After this it undergoes, ATP-dependent oligomerisation in which it binds to lower affinity dnaA boxes. This produces an oligomeric nucleoprotein complex.

Front 35

What is the sequence of a dnaA box?

Back 35

5' TTATCCACA 3'

Front 36

What are found downstream from dnaA boxes in OriC?

Back 36

dnaA trios are found downstream from dnaA boxes. These are 3 nucleotide sequences with an adenine every 3 bases (G/A-A-T).

These dnaA trios are bound by the AAA+ domain of dnaA (when they are split to ssDNA due to torsion produce from dnaA oligomerisation)

Front 37

How many dnaA boxes are there usually at OriC? And which have the most severe effect on replication when removed?

Back 37

7

The 6th and 7th dnaA boxes have most serious effect on replication when deleted. These boxes bind the dnaA which interacts with the dnaA trios in order to separate them to ssDNA, so without these dnaA does not allow loading of the dnaB helicase so replication cannot occur.

Front 38

What is found between dnaA boxes and dnaA trios?

Back 38

There is a GC-rich sequence.

Front 39

After dnaA trios have been separated to ssDNA and bound by dnaA what happens next in replication initiation?

Back 39

3-6 molecules of dnaC are used to form dimers which interact with dnaB (in the presence of ATP)

dnaC dimers form an asymmetrical dumbbell shape which gives them a twist of 50 degrees. This twist allows them to make extensive contact with dnaB which allows it to be split apart.

dnaC then hydrolyses ATP and dissociates.

dnaB is then loaded onto the DNA. It then moves 5' to 3' along the DNA, separating the DNA into single strands.

Front 40

What is the function of single-stranded binding protein in prokaryotes?

Back 40

It binds to single-stranded DNA as it is unwound in replication in order to prevent it from forming complex secondary structures .e.g. hairpins.

Front 41

How long are the RNA primers created by DNA primase/dnaG?

Back 41

~5bp

Front 42

What is the function of the Beta subunit of DNA polymerase?

Back 42

It acts as a sliding clamp which binds DNA polymerase and clamps double-stranded DNA to keep them together. This improves both speed and processivity.

Front 43

What are the improvements in speed and processivity of DNA polymerase when it has the beta sliding clamp compared to when it doesn't?

Back 43

Its speed increases from 20bp/second to 750bp/second.

It can stay on DNA for up to 50kb in vitro compared to 10bp without the clamp.

Front 44

What is the function of the AAA+ clamp loader?

Back 44

This protein loads the beta sliding clamp on to DNA while ATP bound before hydrolysing ATP and dissociating from the clamp.

It also anchors the two replisomes together to ensure that replication of both strands is co-ordinated.

Front 45

What is the function of the alpha subunit of DNA polymerase III?

Back 45

It is the polymerase subunit.

Front 46

What is the function of the epsilon subunit of DNA polymerase III?

Back 46

It is the exonuclease subunit.

Front 47

What is the function of the theta subunit in DNA polymerase III?

Back 47

This subunit is not essential to DNA polymerase III activity but it provides stability to the epsilon subunit, improving its exonuclease activity.

Front 48

Which domains are found in the alpha subunit of DNA polymerase III?

Back 48

A PHP domain.

Palm, thumb and finger domains.

A beta binding domain.

A C-terminal domain.

Front 49

What is the function of the PHP domain in the alpha subunit of DNA polymerase III?

Back 49

It is effectively useless and is just a relic of evolution as it appears to have formerly been able to co-ordinate metal ions, likely to provide it with catalytic exonuclease activity, but evidently the epsilon subunit was more effective so the PHP domain lost its function.

Front 50

How long is DNA polymerase III?

Back 50

Around 1200 amino acids long.

Front 51

Why have we gained most of our structural information from DNA polymerase I as opposed to the more important DNA polymerase III?

Back 51

DNA polymerase I is only made from one polypeptide so it is far easier to study than DNA polymerase III.

But it still has the key architectural motifs for the finger, palm and thumb domains and an exonuclease domain.

Front 52

What are the functions of the finger, thumb and palm domains in DNA polymerase?

Back 52

The finger domain binds ssDNA and dNTPs.

The thumb domain binds dsDNA.

The palm is the active site.

Front 53

How does DNA polymerase I ensure that the incoming nucleotide is complementary to the template strand?

Back 53

The incoming dNTP binds the finger domain and induces a conformational change which, if it is complementary to the template strand, will allow it to pass through to the palm where its addition to the chain can be catalysed.

Front 54

Explain the function of the magnesium ions in the active site of DNA polymerase?

Back 54

Nucleophilic attack of the 3'OH on the 5' phosphate can only occur within 2.5 angstroms. To allow these negative charges to occur in such close proximity, the magnesium ions shield the charges.

These magnesium ions are each co-ordinated by 6 bonds, 2 of these are conserved aspartate residues. This hexa-co-ordination produces the correct geometry needed for catalysis.

One of the Mg2+ ions activates the 3'OH nucleophile and the other stabilises the beta-gamma pyrophosphate leaving group.

Front 55

Why are topoisomerases needed in DNA replication?

Back 55

As DNA is unwind by helicases, this produces torsional stress downstream from the replication fork which must be relieved in order that the replisome can pass through it.

Front 56

How do Top1B topoisomerase I enzymes work?

Back 56

They use a tyrosine residue in their active site to bind a 5' phosphate in DNA. This covalent bond formation causes the phosphodiester backbone to break. The DNA can then rotate in order to relieve the supercoiling.

The energy stored in the phosphotyrosine bond can be used to reform the phosphodiester bond, thus returning the topoisomerase back to its original state.

Front 57

How do Top1A topoisomerase I enzymes work?

Back 57

Top1A toposiomerases can melt DNA locally. They then bind the ssDNA and cleave the other strand, they then pass the uncleaved strand through the cleaved strand before re-ligating the cleaved strand to reform the DNA.

Front 58

What enzymes are used when DNA supercoiling becomes particularly severe? And how does it work?

Back 58

DNA gyrase/Topoisomerase II is used.

It binds one dsDNA segment, it then binds another.

It cleaves the first dsDNA segment and passes the second one through and out the bottom of a gap in the protein. It then re-ligates the first dsDNA segment.

Front 59

Give 4 things which can cause replisome collapse

Back 59

DNA-bound proteins

Damaged DNA

Collision with transcription machinery (leading to R loop formation)

Sequence at risk motifs

Front 60

What are sequence at risk motifs?

Back 60

These are long repeating sequences which, when unwound during replication, can fold back on themselves to form hairpin loops or other secondary structures.

Front 61

How does the cell attempt to avoid collision of the replisome with transcription machinery?

Back 61

Highly-transcribed genes are directed in the same direction as replication fork progression as head-to-tail collisions are far better than head-on collisions.

Front 62

What is the process of replication of recover of the replisome?

Back 62

PriA recognises and binds stalled replication forks. It then uses its 3' to 5' helicase activity to unwind DNA.

PriB then binds PriA and recruits a helicase loader called dnaT.

PriA-PriB-dnaT binds single-stranded DNA to prevent single-stranded DNA binding protein from binding.

This complex then loads dnaB-dnaC onto ssDNA.

Then all of the proteins except dnaB dissociate.

Front 63

What marks the end of replication in E.coli?

Back 63

Tus-ter complexes. Where a protein called Tus binds a 23bp termination site called a ter site.

Front 64

How do Tus-ter end replication in E.coli?

Back 64

In one conformation the replication fork can displace the tus protein. But in the opposite conformation, replication fork contact with the tus protein causes the tus protein to form an incredibly tight complex with DNA which cannot be displaced by the replisome.

Front 65

What is the function of eukaryotic polymerase alpha?

Back 65

It is a lagging strand primase and polymerase, meaning that it has both DNA polymerase and RNA polymerase activity.

It synthesises an RNA primer between 7 and 10bp long. It then extends this primer by 20-30bp with DNA.

Front 66

What are the roles of the 4 subunits in polymerase alpha?

Back 66

P48 = Produces the RNA primer

P58 = Stabilises the P48 subunits and allows it to interact with P180

P180 = The DNA polymerase subunit

P86 = A phosphorylated regulatory subunit

Front 67

What is the function of eukaryotic polymerase delta?

Back 67

This is a lagging strand polymerase

Front 68

What is the function of eukaryotic polymerase epsilon?

Back 68

It is a leading strand polymerase.

Front 69

Which polymerases in eukaryotes associate with proliferating cell nuclear antigen and what is its structure?

Back 69

Polymerase delta and polymerase epsilon associate with proliferating nuclear cell antigen which is the analogue of the beta sliding clamp in prokaryotes.

Front 70

The clamp structure is conserved in prokaryotes and eukaryotes, what is the main difference between them?

Back 70

The clamp is a dimer in prokaryotes and a trimer in eukaryotes.

Front 71

How does replication terminate in eukaryotes?

Back 71

Two replication forks converge and cause SCF^Dia2, an E3 ligase, to ubiquitinate Mcm7. This ubiquitination causes Cdc48 recruitment which is an ATPase required for CMG helicase disassembly.

Front 72

Which proteins are used to load Mcm2-7?

Back 72

Cdc6 and Cdt1

Front 73

In what direction does the Mcm2-7 helicase translocate along DNA?

Back 73

3' to 5'

Front 74

What are the 3 components of an active eukaryotic helicase?

Back 74

Cdc45

Mcm2-7

GINS

Front 75

What is the process of CMG helicase formation?

Back 75

Mcm2-7 is phosphorylated by DDK which drives CM formation.

Cdk then phosphorylates Sld2 and Sld3 which drive CMG formation.

Mcm10 then activates the helicase.

Front 76

How does Mcm10 activate the CMG helicase?

Back 76

Mcm10 binds Mcm2 and produces a conformational change which stabilises the interactions of Cdc45 and GINS with Mcm2-7. This stability then allows the complex to begin unwinding the DNA.

Front 77

During what parts of the cell cycle is CMG helicase loaded and activated?

Back 77

During G1 there are low Cdk levels so helicases are loaded but not activated. Whereas, in S phase, there are high Cdk levels which means there is helicase activation but not helicase loading.

Front 78

What enzyme produces telomeres?

Back 78

Telomeres are produced by an enzyme called telomerase which is a specialist ribonucleoprotein reverse transcriptase. It contains an RNA template which encodes a hexanucleotide sequence which is added at chromosome ends.

Front 79

What is the hexanucleotide repeat sequence of a telomere?

Back 79

TTAGGG

Front 80

How do telomeres form and how are they kept stable?

Back 80

The free 3' tail loops back on itself and displaces one of the strands in the double-stranded DNA to form a complementary interaction. This is called a T-loop.

This telomere is kept stable by a protein complex called shelterin

Front 81

How does shelterin cause the cell to differentiate between dsDNA breaks and chromosome ends?

Back 81

Shelterin binds ATM kinase at a region containing S1981 which is the residue which is autophosphorylated when ATM is activated by DNA damage. This means that ATM kinase activation is prevented, so proteins like P53 also remain inactive.

Front 82

What are the natural functions of recombination?

Back 82

It is used to repair double-stranded DNA breaks.

Front 83

What are the synthetic functions of recombination?

Back 83

It can be used to integrate viral genomes into host chromosomes.

It can be used to create genetic diversity in meiosis.

It can be used to generate antibody diversity.

Front 84

How do single-strand breaks and collapsed replication forks lead to double-stranded breaks?

Back 84

A single-stranded break prevents passage of replication machinery, so when this strand is replicated, it will only replicate as far as the single-strand break, thus producing a double-strand break.

Front 85

How do homologous chromosomes align at the site of a double-stranded DNA break?

Back 85

RecA aligns the DNA sequences by binding double-stranded DNA and allowing the undamaged single-stranded DNA to slide through its single-stranded DNA binding site until it reaches a region of complementarity at which the strength of the interaction prevents further sliding.

E C Greene // Journal of Biological Chemistry

Front 86

What is the function of RecB?

Back 86

It is a 3' to 5' helicase and a multifunctional nuclease.

Front 87

What is the function of RecC?

Back 87

Recognises crossover hotspot instigator (chi) site

Front 88

What is the sequence of a Chi site?

Back 88

CGTGGTGG

Front 89

What is the function of RecD in recombination?

Back 89

RecD is a 5' to 3' helicase.

Front 90

What is the process from RecBCD binding a double-stranded break to RecA binding?

Back 90

Rec BCD uses its helicase functions to unwind DNA, this DNA is degraded by RecB as the complex moves along.

When Rec C recognises a Chi site, this induces a conformational change in the complex which causes greater degradation of the 3' to 5' strand (as there are two exit tunnels in Rec B, one passes via the RecB active site and one doesn't so after hitting the Chi site, the 5' to 3' is passed through the tunnel away from the RecB active site)

Front 91

What happens after RecA is loaded onto the 3' tail of a processed double-stranded DNA break?

Back 91

RecA undergoes ATP-dependent oligomerisation to form filaments.

Each RecA monomer interacts with 3 nucleotides.

RecA's primary binding site binds ssDNA, its secondary binding site binds dsDNA. It breaks the strands of dsDNA apart and spools in the single-stranded DNA to test for complementarity.

If 15 bases of complementarity are present then strand exchange can occur.

Front 92

Give basic details about RecA filaments

Back 92

RecA filaments have a pitch of 83 angstroms.

There are 6 monomers per turn.

Front 93

Branched DNA structures are common recombination intermediates, what is the process of extending or shortening these known as?

Back 93

Branch migration

Front 94

How are Holliday junctions extended and resolved?

Back 94

RuvA forms a tetramer where each monomer binds one strand in the complex.

2 RuvB, a hexameric ring molecular motor, bind the RuvA tetramer.

After this complex has extended the branch, RuvC, a nuclease, comes and cleaves the junction to resolve it.

It can binds in one of two orientations meaning that it can produce horizontal, vertical or horizontal and vertical cleavage.

Front 95

In recombination what are spliced and patched products?

Back 95

Spliced products = Extensive recombination has occurred where long stretches of DNA have been swapped.

Patched products = Small amounts of recombination where only short stretches of DNA have been swapped.

Front 96

Viral recombination occurs via phage integrases, what family are these enzymes a part of?

Back 96

The tyrosine recombinase family

Front 97

How do tyrosine recombinases work?

Back 97

Tyrosine recombinases function as tetramers (R1, R2, R3 and R4).

R1 and R3 cut the top strand (5' to 3') using an active site tyrosine to bind the 5' phosphate of DNA, thus breaking the phosphodiester backbone.

While this DNA-tyrosine intermediate is present, the top strands are exchanged and they form a Holliday junction.

R2 and R4 then cut and exchange the bottom strands, resolving the Holliday junction and giving recombined products.

Front 98

What is the structure of the recombination sites in viruses?

Back 98

There is a phage attachment site (P O P')

and a bacterial attachment site (B O B')

P, P', B and B' are the recombination recognition sites and O is the actual crossover site.

Front 99

What is the eukaryotic complex which recognises double-strand breaks in homologous recombination?

Back 99

The MRX complex (2 Mre11 molecules, 2 Rad50 molecules and 1 Xrs2 molecule)

Front 100

What occurs in eukaryotic homologous recombination after MRX caps the dsDNA break?

Back 100

Sae2, a nuclease, creates a nick in the bottom strand 20nts from the 5' end.

Exo 1 (5' to 3' endonuclease), Sgs1 (3' to 5' helicase) and dna2(ssDNA endonuclease/helicase) function to complete resection.

Front 101

Which complex inhibits the Exo1-Sgs1-dna2 complex in eukaryotic homologous recombination?

Back 101

Ku70-Ku80

Front 102

After resection in eukaryotic homologous recombination, RPA binds the ssDNA, then what happens?

Back 102

RPA (single-stranded binding protein homologue) is replaced by Rad51 which is recruited by Rad52.

Rad51 is then bound by Rad55 and Rad57 which stabilise the nucleoprotein filament.

Rad54 then interacts with Rad51 to promote chromatin remodelling, DNA unwinding and strand annealing.

Front 103

What disease is caused by a lack of Sgs1?

Back 103

Bloom's syndrome

Front 104

What are the symptoms of Bloom's syndrome?

Back 104

Excess recombination leads to genomic instability. This leads to excess sun sensitivity causing a butterfly-shaped rash on exposed areas.

Individuals have limited fat as gastro-oesophageal reflux removes the desire to eat.

They are also susceptible to early-onset diabetes and some cancers.

Front 105

Which eukaryotic complexes produces crossover products?

Back 105

Mus81-Mms4 is a nuclease produces asymmetric crossover products (one cleavage vertical and one horiztonal)

Front 106

How do Srs2 (a helicase), BLM (3' to 5' helicase), Fml1 (a helicase) and RTEL1 (a helicase) resolve recombination intermediates?

Back 106

D-loops begin to form and be copied but RTEL1 interrupts the loop and by this time there is enough overlap in the strand that the DNA can act as a template without need for crossover.

Front 107

What enzyme can be used to cleave double Holliday junctions horizontally or vertically to produce patched or spliced products?

Back 107

Yen1

Front 108

How can Sgs1-TopIIIalpha-Rmi1 be used to resolve double Holliday junctions?

Back 108

Sgs1 is a 3' to 5' helicase used in branch migration.

Topoisomerase III alpha then rotates the strands in the junction after DNA synthesis, thus removing the D-loop and leaving non-crossover products.

Rmi1 appears to join to Sgs1 and TopIIIalpha to improve their catalytic activity.

I D Hickson // Cell Cycle

Front 109

In recombination during antibody formation, what sequences are recognised by RAG1 and RAG2?

Back 109

RAG1 and RAG2 recognise a conserved 7-mer, a 12bp spacer and a 9-mer at the 3' end of the V segment.

They also recognise a conserved 7-mer, a 23bp spacer and a conserved 9-mer at the 5' of the J segment.

Front 110

How does recombination occur in antibody production?

Back 110

RAG1 and RAG2 loop the 2 segments to which they are connected to be next to each other. They then cleave directly between the recombination signal sequence-gene segment border.

The recombination signal sequence then has double-stranded breaks, which are ligated to form the signal joint.

The gene segment DNA forms hairpin loops which are nicked by a complex of DNA-dependent protein kinase, artemis, Ku, XRCC4 and DNA ligase I.

Terminal deoxynucleotidyl transferase then adds more bases to this DNA and the gene segments are ligated by DNA ligase.

Front 111

How does Classic NHEJ occur in eukaryotes?

Back 111

Ku70-Ku80 bind the double-strand breaks to cap them.

53BP1 binds one side of the break, Mre11 binds the other side of the break.

Artemis then binds, one molecule to either side of the break.

XRCC4 and DNA ligase IV then reseal the break with minimum damage to the genome.

Front 112

How does alternative NHEJ occur in eukaryotes?

Back 112

PARP1 binds either side of a double-stranded DNA break.

It recruits the Mre11 complex.

CtIP then binds this complex and guides the strands to a point of homology before XRCC4-DNA ligase IV seals the gap.

This point of small amounts of homology may not be at the end of strands and so some DNA may be cleaved off and lost.

Front 113

How does recombination occur in eukaryotes during meiosis?

Back 113

Spo11 forms a dimer that cuts DNA using a tyrosine group in its active site to form a covalent bond with a 5' phosphate. This produces a dsDNA break.

This permits loading of the MRN/MRX complex and then this allows normal recombination to occur using Sae2, ExoI etc.

Front 114

How many hotspots are there for meiotic recombination per cell?

Back 114

300

Front 115

What are the usual characteristics of recombination hotspots?

Back 115

They are usually flanked by "coldspots" which are areas of lower than average recombination.

They are around 100-500bp (in S.cerevisiae, 1-2kb in humans) long (the regions in which dsDNA breaks occur)

They are thought to be position-specific as deleting these 100-500bp long sequences does not change the amount of recombination in this position of the DNA.

Also they are frequently repeating sequences or retroviral relics.

Front 116

Which protein is required for interhomologous chromosome recombination?

Back 116

Dmc1, an octameric ring structure, with 45% sequence homology to RecA.

Front 117

Spontaneous loss of a purine or pyrimidine base creates what type of site?

Back 117

An abasic site.

Front 118

Give 4 examples of deamination and what their products pair with

Back 118

Cytosine to Uracil (Binds adenine)

Guanine to Xanthine (Cannot pair to any base)

5-methyl Cytosine to Thymine (Binds adenine)

Adenine to hypoxanthine (Binds guanine)

Front 119

Give an example of how reactive oxygen species can damage DNA

Back 119

Reactive oxygen species can convert thymine to thymine glycol

Front 120

How can alkylation affect DNA?

Back 120

Alkylation can result in the production of 3-methyladenine or 7-methyladenine with S-adenosylmethionine as a co-factor.

Front 121

Photodamage can cause pyrimidine dimers to form, what is the relative occurrence of different pyrimidine dimers?

Back 121

T-T > T-C > C-T > C-C

Front 122

By how much is DNA bent and unwound when thymine dimers form?

Back 122

Bent by 30 degrees, unwound by 9 degrees.

Front 123

Photodamage can cause pyrimidine (6-4) pyrimidone photoproducts to form, what is the relative occurrence of these photoproducts?

Back 123

T-C >> C-C > T-T > C-T

Front 124

What degree of bending is provoked in the DNA helix by pyrimidine (6-4) pyrimidone photoproducts?

Back 124

44 degrees

Front 125

Other than photoproducts, give 4 other means of endogenous DNA damage?

Back 125

Replication errors

Intra/Inter-strand cross-linking

DNA-Protein cross-links

Strand breaks

Front 126

Give 4 types of replication error

Back 126

Insertion of the wrong nucleotide

Insertion of a damaged nucleotide

Polymerase slippage

Base misalignment

Front 127

How does polymerase slippage occur?

Back 127

DNA polymerase is replicating a repeating sequence. The nascent strand dissociates from the template strand and the DNA polymerase.

It then uses one of its later repeats to bind an earlier repeat on the template strand. This forms a loop of useless DNA.

This can cause insertion mutations when this strand is later replicated.

E Viguera // EMBO

Front 128

What are the 5 ways in which ionising radiation can damage DNA?

Back 128

It can cause ssDNA breaks

It can cause dsDNA breaks

It can cause oxidation of bases (by producing oxygen radicals)

It can cause deletions

It can produce gross chromosomal abnormalities.

Front 129

How can UV damage DNA?

Back 129

It can cause cross-linking of bases leading to misreading of DNA.

Front 130

How can free radicals damage DNA?

Back 130

They can deaminate cytosine to uracil.

Front 131

How can bulky adducts damage DNA?

Back 131

Bulky adducts like aflatoxin or ethidium bromide can integrate into DNA and cause distortion of the DNA structure, leading to misreading and strand breaks.

Front 132

How many purines and pyrimidines are spontaneously lost each day per cell?

Back 132

18,000 purines and 600 pyrimidines are lost each day.

Front 133

How many cytosine molecules are deaminated per cell per day?

Back 133

100-500

Front 134

How many adenine molecules are alkylated each day per cell?

Back 134

1200

Front 135

How does DNA photolyase repair cyclobutane dimers?

Back 135

DNA photolyase binds the cyclobutane dimer.

Blue light is absorbed by methenyl tetrahydrofolate (MTHF) which acts as a photantenna. This excites an electron which it then passes to FAD (the photocatalyst)

Excited FAD donates the electron to the CPD which causes electron rearrangement and restoration of the native DNA conformation before the electron is donated back to FAD.

Front 136

What can replace methenyl tetrahydrofolate (MTHF) as a photoantenna in other organisms?

Back 136

8-hydroxy-5-deazeflavin (8-HDF)

Front 137

What is the optimum wavelength absorbed by MTHF when it is bound to DNA photolyase?

Back 137

377-410nm

Front 138

How does DNA photolyase bind DNA?

Back 138

It has a cavity on its surface lined with positively-charged residues which allow it to bind the negatively-charged phosphate backbone of DNA. (K463, R507, K517)

S Weber // BBA-Bioenergetics

Front 139

What is the function of AlkB in repairing DNA damage?

Back 139

It converts 3-methylcytosine or 1-methyladenine into an intermediate which then degrades to form just cytosine or adenine.

In the production of this intermediate, alpha ketoglutarate and oxygen are required and succinate and CO2 are produced.

This means that this is an oxidative decarboxylation reaction.

Front 140

How many AlkBs do humans have and which is deemed to be the most important?

Back 140

Humans have 9 AlkBs.

AlkBH2 is deemed to be most important as it can demethylate both 3-methylcytosine and 1-methyladenine.

Front 141

How is O6-methylguanine converted back to guanine?

Back 141

O6-methylguanine methyltransferase removes the methyl group from the guanine and transfers it to an active site cysteine residue.

Front 142

What is the effect of O6-methylguanine methyltransferase removing a methyl group from guanine on signalling?

Back 142

O6-methylguanine methyltransferase is a suicide enzyme. However, in its methylated state, it acts as a signalling protein to upregulate O6-MGT production.

Front 143

In what organism are 6-4 photolyases not present?

Back 143

E.coli

Front 144

How can DNA photolyase be used to repair pyrimidine (6-4) photoproducts?

Back 144

DNA photolyase uses a conserved active site histidine (H364/H365 depending on the organism) to donate a proton to C5 of the 5' nucleotide in the lesion as well as still donating the electron.

This proton is then transferred to N3 of the same molecule. This leads to bond rearrangement which repairs the lesion.

The proton and electron are then donated back to FAD.

D Zhong // Nature

Front 145

What percentage of mutations remaining after DNA synthesis and exonuclease activity are removed by the mismatch repair system?

Back 145

99.9%

Front 146

What 4 types of DNA damage can be repaired by the mismatch repair system?

Back 146

Misincorporated bases

Small insertions

Small deletions

Misalignments in microsatellites caused by polymerase slippage

Front 147

How does mismatch repair machinery recognise which strand is the new strand?

Back 147

Adenine residues on the old strand in the sequence GATC are methylated by DAM methylase immediately after DNA replication.

Front 148

What is the basic process of mismatch repair in E.coli?

Back 148

MutS recognises and binds the mismatch as a homodimer. A different domain from each monomer, with different function, binds the mismatch, effectively making it seem like a heterodimer has bound.

MutL binds MutS to stabilise the dimer.

MutH is then recruited and it recognises hemimethylated DNA, it produces a nick direclt opposite the methyl group.

MutL then recruits UvrD which is a helicase and single-strand exonuclease. It binds at the nick and unwinds the duplex. Then Exo1 or RecJ cleaves the nicked strand.

DNA polymerase III then fills in the gap which is then re-ligated by DNA ligase.

Front 149

What does the new model of mismatch repair in E.coli propose about MutS function?

Back 149

It proposes that MutS binds and clamps a mismatch, it then binds ATP to make it a sliding clamp.

MutL is recruited and MutS-MutL slides slowlt along DNA . MutL then recruits MutH.

MutL and MutH then detach and search for hemimethylated DNA.

Front 150

What is the human MutS alpha complex made of?

Back 150

MSH2-MSH6 (80-90%)

Front 151

What is the human MutS beta complex made of?

Back 151

MSH2-MSH3

Front 152

What is the human MutL alpha complex made from?

Back 152

MLH1-PMS2

Front 153

What is the human MutL beta complex made from?

Back 153

MLH1-PMS1

Front 154

What is the function of exonuclease I in mismatch repair in eukaryotes?

Back 154

It is a 5' to 3' exonuclease.

Front 155

What is the function of proliferating cell nuclear antigen in eukaryotes?

Back 155

It is homologous to the sliding clamp in prokaryotes.

Front 156

What is the function of replication factor C in eukaryotes?

Back 156

It is a 5 subunit complex which acts as a clamp loader.

Front 157

What is the function of MutS alpha in eukaryotes?

Back 157

It targets single base pair mismatches or small insertion/deletion loops.

Front 158

What is the function of MutS beta in eukaryotes?

Back 158

It targets larger insertion of deletion loops.

Front 159

What is the major form of MutL in eukaryotes?

Back 159

MutL alpha

Front 160

What is used to ensure that it is always the nicked strand that is repaired in eukaryotes?

Back 160

The replication fork is used to identify the new strand.

Front 161

Exonuclease I is a 5' to 3' exonuclease, so it can easily remove a mismatch if the nick is 5' of the mismatch, but how can it function where the nick is 3' of the mismatch?

Back 161

MutL alpha's PMS2 subunit is an endonuclease which means it introduces nicks into the mismatched strand either side of the mismatch. This means there is a 5' nick on to which exonuclease I can load.

Front 162

What disease can be caused by defective mismatch repair?

Back 162

Hereditary non-polyposis colon cancer.

Front 163

Hereditary non-polyposis colon cancer makes up what percentage of colon cancer in the Western world?

Back 163

2-4%

Front 164

What is the median age at which people with defects in mismatch repair develop hereditary non-polyposis colon cancer?

Back 164

40

Front 165

What is the percentage risk of HNPCC by the age of 70 for an individual with defects in mismatch repair?

Back 165

80%

Front 166

What are the two main mismatch repair genes which are mutated in causing HNPCC?

Back 166

MSH2 = 40%

MLH1 = 40%

These mutations are autosomal dominant.

Front 167

Individuals with mismatch repair defects very commonly get HNPCC, to what other types of cancer are they susceptible?

Back 167

Endometrium

Ovary

Stomach

Epithelial tumours

Front 168

What type of genomic instability is present in HNPCC and 15-20% of other cancers?

Back 168

Microsatellite instability.

This is where deletion of parts of microsatellites alters gene regulation and genome stability. This is coupled with MLH1 promoter methylation to prevent repair of this instability.

A Goel // Gastroenterology

Front 169

There are multiple base excision repair pathways, but what are the common features between all of them?

Back 169

They all use a DNA glycosylase to remove incorrect bases.

A nick is produced upstream from the abasic site using an AP endonuclease.

The strand is extended from the 3'OH by a DNA polymerase which also cleaves 3' of the abasic site.

Front 170

Describe the process of short patch base excision repair

Back 170

DNA glycosylase hydrolyses the glycosidic bond between the sugar and the base to remove the base.

AP endonuclease recognises the abasic site and cleave 5' of the abasic site.

DNA polymerase Beta then uses its phosphodiesterase function to cleave 3' of the abasic site. It then uses its polymerase function to fill the gap.

DNA ligase then seals the nick.

Front 171

What is AP lyase function in regards to DNA glycosylases?

Back 171

Some DNA glycosylases have dual-function, other than cleaving off a base, they can also use a beta-elimination reaction to cleave 3' of the abasic site.

Front 172

In what form do DNA glycosylases function?

Back 172

They function in monomeric form without any co-factor or divalent cation.

Front 173

How do DNA glycosylases work?

Back 173

They locate lesions, bind it to induce DNA bending.

This bending occurs until the base pairing is interrupted and flips out into the enzyme pocket.

Front 174

What happens if individuals have mutations in the MutY gene?

Back 174

This gene is involved in the repair of 8-oxoguanine lesions.

If mutations are present this leads to MutYH-associated colorectal cancer which makes up 1% of all colorectal cancer.

These mutations also lead to mutations forming in the adenomatous polyposis coli (APC) gene which controls proliferation of colon cells. This therefore leads to the individual's colon becoming littered with adenomatous polyps.

Front 175

What is the process of long patch base excision repair?

Back 175

DNA glycosylase produces an abasic site.

AP endonuclease nicks 5' to the lesion.

DNA polymerase delta or epsilon join on the free 3' OH and begin to extend the DNA, making a flap where the incorrect DNA has been displaced.

DNase IV then cleaves the flap (Rad27 in yeast)

DNA ligase I then seals the gap.

Front 176

How does Uracil DNA glycosylase cause uracial to flip out of DNA into its active site?

Back 176

This enzyme uses electrostatic interactions between residues in its active site and the phosphate backbone to interact with DNA.

It inserts residue 272 into the minor groove of DNA which warps DNA structure.

When these two factors are combined with compression of the DNA caused by the presence of uracil cause the DNA to flip out into the glycosylase active site.

G Slupphaug // Nature

Front 177

Which process is more flexible: base excision repair or nucleotide excision repair?

Back 177

Nucleotide excision repair; it can repair UV-induced photoproducts and bulky adducts which cannot be repaired by DNA.

Front 178

The process of nucleotide excision repair was discovered in individuals with which disease?

Back 178

Xeroderma pigmentosa

Front 179

What is the beginning of the process of nucleotide excision repair in E.coli (up to UvrB)?

Back 179

2 molecules of UvrA and 2 molecules of UvrB bind damaged DNA as a central UvrA dimer with flanking UvrB monomers.

UvrA hydrolyses ATP and dissociates, stabilising the interaction of UvrB with DNA. UvrB also bends the DNA at this point.

Front 180

What is the process of nucleotide excision repair in E.coli (after UvrB)?

Back 180

After UvrB has induced a bend in DNA UvrC binds next to it. This binding activates UvrC to nick the DNA around 4 nucleotide 3' of the DNA damage.

It then nicks around 7 nucleotide 5' of the damage. (These nicking steps require ATP binding but not hydrolysis)

UvrD uses energy from ATP hydrolysis to displace the damage-containing sequence, it displaces UvrC at the same time.

DNA polymerase I then fills the gap and displaces UvrB at the same time.

DNA ligase then seals the gap.

Front 181

How can NER be used to repair cross-linked DNA in E.coli?

Back 181

UvrABC cleaves the leading strand either side of the cross-link.

Exonuclease I then produces a ssDNA region 3' of the cross-link.

RecA then recombines this strand to produce the correct sequence, displacing the cross-linked section.

UvrABC then cleaves the other strand either side of the cross link so that the cross-link is removed.

DNA polymerase then fills the gap.

Freidberg // DNA Repair and Mutagenesis

Front 182

What are the initial stages in Global genomic nucleotide excision repair in eukaryotes?

Back 182

An XPC-hHR23b heterodimer recognises and binds damaged DNA. This binding further distorts DNA.

TFIIH then binds the DNA. This permits XPA to access the damaged region which it unwinds.

XPD then furhter unwinds the DNA allowing RPA to bind.

CAK then dissociates from TFIIH

Front 183

What determines the recruitment of XPC-hHR23b in global genomic nucleotide excision repair?

Back 183

The greater the distortion, the more readily this heterodimer is recruited. So it is more readily recruited to pyrimidine (6-4) pyrimidone photoproducts than cyclobutane dimers.

Front 184

What are the initial stages in transcription-coupled nucleotide excision repair?

Back 184

DNA damage is recognised by stalled RNA polymerase II. After stalling, RNA polymerase II recruits TFIIH via CSA and CSB.

TFIIH then unwinds a 20-30 nucleotide stretch.

RPA and XPA are recruited.

CAK then dissociates.

Front 185

After the initial stages of global genomic NER and transcription-coupled NER?

Back 185

XPG and XPF-ERCC1 are recruited.

XPG cuts 2-8 nucleotides 3' of the damage.

XPF-ERCC1 cuts 15-24 nucleotides 5' of the damage.

Replication factor C, PCNA and DNA polymerase delta or epsilon bind the 3'OH and fill the gap.

DNA ligase I then seals the gap.

Front 186

Cyclobutane dimer recognition by XPC-hHR23b is dependent on what?

Back 186

This recognition is dependent on DDB1-XPE which make an E3 ligase.

They monoubiquitinate XPC in order to increase its affinity for the lesion.

They also monoubiquitinate histones to remodel them.

They also monoubiqtuinate XPE causing it to dissociate from DNA.

Front 187

What percentage of individuals suffering from Xeroderma pigmentosa have mutations in XPA-XPG genes?

Back 187

75%

Front 188

What are the effects of having Xeroderma pigmentosa?

Back 188

There is hypersensitivity to light which leads to a high incidence of UV-related cancers.

At 18 months, 50% of children will have developed freckle-like lesions with erythema or increased pigmentation.

Benign skin tumours are very common but skin cancer has a median onset age of 8.

This condition increases the likelihood of neurological conditions and other cancers.

Front 189

What mutations cause Cockayne's syndrome?

Back 189

Mutations in CSA and CSB (predominantly CSB)

Front 190

What are the effects of Cockayne's syndrome?

Back 190

Some sufferers are light sensitive.

They suffer neuronal abnormalities.

Premature ageing of some tissue

Facial and limb abnormalities

Short stature

Early death due to neurodegeneration

Front 191

What mutations cause thriothiodystrophy?

Back 191

Mutations are in TTD-A, XPB-TTD and XPD-TTD.

Front 192

What are the effects of trichothiodystrophy?

Back 192

Some individuals are light-sensitive.

Premature ageing of some tissues

Facial abnormalities

Short stature

Ichthyosis

Sulfur-deficient brittle hair.

Front 193

How can double-strand breaks form?

Back 193

Ionising radiation

A nick in the leading strand or lagging strand can cause replication fork collapse

Any other lesion that results in replication fork collapse .e.g. doxorubicin integrates into DNA and causes replication fork collaps.

Topoisomerase I can mulfunction and remain covalently bound to DNA, thus preventing replication fork progression.

Front 194

How many dsDNA breaks form per replication cycle?

Back 194

50

Front 195

How can RecA be used in post-replication recombination repair?

Back 195

When a DNA replication fork encounters damage it moves slowly around it before replication the rest of the strand.

The unreplicated section of this strand is fixed by recombination using homologous chromosomes.

Front 196

How is RecA involved in the SOS response in E.coli?

Back 196

DNA damage activates RecA.

RecA binds the LexA repressor and activates autocatalytic cleavage which causes LexA-regulated gene expression.

11 crucial genes in the SOS response are controlled by the LexA promoter, so they are activated (this includes UvrA and UvrB so NER is enhanced. It also includes RecA)

Front 197

How is DNA polymerase V used in the SOS response in E.coli?

Back 197

DNA polymerase V uses the damaged strand as a template to produce dsDNA. This prevents single-stranded DNA being a target of exonuclease.

P Pham // PNAS

Front 198

Homologous recombination in eukaryotes requires the RAD51 epistasis group. What is an epistasis group?

Back 198

When two mutations are introduced into a cell, if this has a greater effect than either mutation alone then they are not in the same epistasis group.

Whereas, if either mutation or both mutations together has the same effect then the gene are in the same epistasis group.

Front 199

What is RAD51 in eukaryotes homologous to in E.coli?

Back 199

RecA

Front 200

How is BRCA2 involved in homologous recombination in eukaryotes?

Back 200

BRCA2 binding to RAD51 is required for homologous recombination to occur. This was proven by adding a small amino acid sequence from BRCA2's RAD51 binding site and adding it to normal cells. It interferes with the activity of RAD51 by preventing binding of functional BRCA2.

Front 201

What makes up the MRN/MRX complex in eukaryotic homologous recombination?

Back 201

MRN (humans) = Mre11 (A nuclease)

RAD50 (ATPase, zinc finger and coiled coil)

Nbs1 (FHA/BRCT domains)

Xrs (in yeast instead of Nbs1).

Front 202

In homologous recombination in eukaryotes, what caps double-stranded breaks?

Back 202

The MRN complex

Front 203

How does the MRN complex activate ATM kinase after recruiting it in homologous recombination?

Back 203

RAD50 in its ATP-bound, closed conformation binds ATM kinase and induces a conformational change which activates it.

Front 204

What is the function of RAD50 in homologous recombination?

Back 204

It has A and B domains which bind to form an ATPase domain, it uses this domain to interact with DNA.

It also uses its zinc hook domain to interact with the other RAD50 on the other side of the break in order to tether them together.

Front 205

After MRN has been recruited and the 2 RAD50 molecules have tethered a dsDNA break, what happens next in eukaryotic homologous recombination?

Back 205

CtIP is recruited by MRN. It then resects the lagging strand to produce a leading strand 3' ssDNA overhang

This resection is stimulated via interaction wth BRCA1 via an unknown mechanism.

Front 206

After resection, what occurs in eukaryotic homologous recombination?

Back 206

RPA then bind ssDNA.

BRCA2 then mediates RPA replacement with RAD51.

RAD51 finds a homologous sequence to the ssDNA and uses it as a template to rebuild the second strand.

Front 207

What is the effect of null mutations in MRN complex genes?

Back 207

These mutations are embryonically lethal.

Front 208

What are mutations in the Mre11 gene associated with?

Back 208

Ataxia telangiectasia-like disorder

Front 209

What are mutations in the Nbs1 gene associated with?

Back 209

Nijmegen break syndrome

Front 210

How many mutations have been found in the BRCA2 gene, and what is the major effect of these?

Back 210

Over 800.

Most of these mutations increase risk of breast cancer. Mutation of one copy is sufficient for increased risk.

Front 211

What is the main mechanism of dsDNA break repair in mammals?

Back 211

Non-homologous end joining (NHEJ)

Front 212

Is NHEJ present in microorganisms?

Back 212

Yes it is present in unicellular eukaryotes like S.pombe and S.cerevisiae but not prokaryotes like E.coli.

Front 213

In which 3 ways have gene involved in NHEJ been isolated?

Back 213

They were isolated by biochemical fractionation of extracts capable of end joining.

They were found via genetic and biochemical analysis of the proteins involved in V(D)J recombination.

They were found via genetic analysis of yeast, rodent and human cell lines.

Front 214

What is the S.cerevisiae Lig4 gene in humans and what is its product?

Back 214

Lig4

DNA ligase IV

Front 215

What is the S.cerevisiae Lif1 gene in humans and what is its product?

Back 215

XRCC4

XRCC4

Front 216

What is the S.cerevisiae HDF2 gene in humans and what is its product?

Back 216

XRCC5

Ku80

Front 217

What is the S.cerevisiae HDF1 gene in humans and what is its product?

Back 217

XRCC6

Ku70

Front 218

What does the XRCC7 gene produce in humans?

Back 218

DNA-dependent protein kinase

Front 219

What does the Artemis gene produce in humans?

Back 219

Artemis nuclease.

Front 220

What are XRCC genes and how were they discovered?

Back 220

Rodent cells are sensitive to X-rays so human DNA was transferred into rodent cells and the cells were tested for X-ray resistance. From this we found X-ray cross complementing (XRCC) genes.

Front 221

What is Ku and what is its function in NHEJ?

Back 221

It is a heterodimer with ATPase activity. It binds DNA breaks and recruits XRCC4, DNA ligase IV and DNA-dependent protein kinase.

Front 222

What is DNA-dependent protein kinase and what is its function in NHEJ?

Back 222

DNA-dependent protein kinase forms a trimer with Ku and is activated by dsDNA ends. At this point it phosphorylates many substrates .e.g. Artemis.

The DNA-dependent protein kinase molecules on either side of the break autophosphorylate one another and dissociate.

Front 223

What can happen when a dsDNA break is introduced between repeats in a repeating sequence?

Back 223

Resection can occur on opposite strands on either side of the break until the next repeat is reached. These two repeats can then bind one another and the tails produced can be cleaved.

DNA polymerase can then fill in the gaps and DNA ligase can seal the nick.

Front 224

What enzymes are used when a dsDNA break is introduced between repeats in a repeating sequence?

Back 224

CtIP resects opposite strands on either side of the break.

RAD52 binds the ssDNA.

ERCC1 associates with XPF, giving the complex nuclease activity which is used to cleave the tails after the complementary repeat sequences have bound.

J M Stark // Cell

Front 225

Why is single-strand annealing rare in humans?

Back 225

We have large amounts of repeating sequences which are key in gene regulation and chromosome structure and this process is deleterious so it would have very negative impacts on these genes and chromosome structure.

Front 226

What is the function of Artemis in NHEJ?

Back 226

This is a nuclease which degrades DNA so it is suitable for ligation.

Front 227

What is the function of XRCC4 in NHEJ?

Back 227

It recruits DNA ligase IV and polynucleotide kinase as well as DNA polymerase beta.

Front 228

What is the function of polynucleotide kinase?

Back 228

It promotes the replacement of the 5' phosphate to ensure it is not damaged.

S C West // EMBO

Front 229

What occurs when there is abortive ligation of DNA?

Back 229

The 5' phosphate remains adenylated. So to restore the phosphate group to normal, it is processed by aprataxin.

S West // Nature.

Front 230

What is microhomology-mediated end joining (MHMEJ)

Back 230

This is a process in NHEJ-deficient cells where the MRX complex binds a dsDNA break.

Rad52 and Rad54 are then recruited.

Resection then occurs and two sequences with microhomology are brought together and bind.

Any left over tail sequence is cleaved.

DNA ligase III then seals the nick.

Front 231

What is alternative end joining?

Back 231

This is where a polymerase binds the break and encodes microhomology sequences which then allow MHMEJ to occur.

DNA ligase I is crucial in nick sealing.

Front 232

Which 2 complexes inhibit MHMEJ and ALT-EJ?

Back 232

Ku70-Ku80

DNA ligase IV-XRCC4

Front 233

What occurs when there is an ssDNA break in eukaryotes?

Back 233

PARP (Poly-ADP ribose polymerase) detects the break and the ends are processed to leave a 5' phosphate and a 3'OH.

The enzyme that completes this processing depends on the type of damage.

PARP then recruits XRCC1 and DNA ligase III.

Polynucleotide kinase and DNA polymerase beta finish the end processing and fill the gap.

DNA ligase III seals the nick.

Front 234

What can cause Ataxia oculometer apraxia repair I?

Back 234

This comes from mutations in the APTX gene which encodes aprataxin which is involved in end processing in ssDNA repair.

Front 235

What can cause spinocerebellar ataxia with axonal neuropathy?

Back 235

Defects in ssDNA repair, specifically mutations in TDP1 which results in mutant tyrosyl-DNA phosphodiesterase production which means topoisomerase I's bond with DNA cannot be broken, thus leading to DNA breaks.