Lecture 4 - recombination

Question 1

RecQ

Answer 1

Helicase that functions at the interface between DNA replication and recombination to repair damage to replication forks.

Helicase domain of 400 amino acids which includes 7 sequence motifs and an ATP binding site.

Question 2

RQC

Answer 2

RecQ-C-terminal
A domain unique to RecQ that consists of a zinc binding domain and a winged helix involved in replication fork recognition.

The HRDC (helicase-and-RNase D-C-terminal) domain is found in RecQ and is involved in DNA binding.

Question 3

What can progression of replication forks be inhibited by?

And what acts to disrupt these

Answer 3

Secondary motifs e.g. hair pins or G quadruplexes

RecQ disrupts these to allow for smooth progression

Question 4

What direction does RecQ helicase move in and what is its function?

Answer 4

RecQ helicase unwinds DNA with a 3’-5’ polarity
Catalyse and get rid of Holliday junction branch migration, fork regression
Unwind DNA loops, triple helices, hairpins, G quadruplex DNA

Question 5

RecQ helicase and TopoIII

Answer 5

From a dissolvasome complex for untangling intertwined structures that arise during DNA replication or repair

Question 6

How do topoisomerases relieve torsional stress?

Answer 6

Break down the DNA backbone in one or both strands and then pass intact DNA through the opening before re-sealing the break

Question 7

What can double Holliday junctions be dissolved by?

Answer 7

Formed by double stranded DNA break
Dissolved by RecQ TopoIII

The two Holliday junctions are migrated towards each other to form a hemicatanane.
The molecules are untabgled by TopoIII.
This leads to non-crossover products and promotes gene stability

Question 8

Describe how RecQ and TopoIII work together to fix double Holliday junctions.

Answer 8

RecQ pushes the Holliday junctions together
RecQ and Topoisomerase III work together to cut one of the strands and untangle this
RecQ and Topoisomerase III completely separate the chromosomes

Conservative method that forms non-crossovers

Question 9

What enzyme seals the nicks?

Answer 9

DNA ligase

Question 10

In gram negative bacteris the branch migration and resolution steps are combined and carried our by which proteins?

Answer 10

RuvABC

Question 11

Function of RuvAB

Answer 11

Perform branch migration

Question 12

Function of RuvC

Answer 12

endonuclease that resolves Holliday junctions

Question 13

Describe RuvA

Answer 13

Forms a tetramer with two channels running across the surface that assembles on Holliday junction DNA.
Specificity is achieved by two negatively charged residues from each monomer forming a protruding pin structure that blocks access of linear dsDNA.
A tetramer of RuvA binds Holliday junction DNA with high specificity.

Question 14

Describe RuvB

Answer 14

A hexamer of RuvB threads onto dsDNA through the centre of the protein rings and acts as a helicase.
ATP hydrolysis drives the pump that translocates along dsDNA. Unlike most other helicases it does not unwind the DNA strand.

Question 15

How do RuvA and RuvB work together?

Answer 15

A complex of RuvA and RuvB unwind Holliday junctions.

Two RuvB monomers target each tetramer block and then full hexamers are assembled.

Question 16

RuvC

Answer 16

The Holliday junction is resolved by the dimeric RuvC endonuclease.
The pair of active sites mediate dual incisions in opposite strands at the junction branch point.
Hydrolysis requires a magnesium to be bound at the active site which is four negatively charged residues.

A dimer of RuvC folds the Holliday junction and cleaves across the point of strand exchange.

Question 17

RuvA targets the _____ and promotes assembly of the _____ _____

Answer 17

junction

RuvB rings

Question 18

RuvC fits on the _____ side of the _____

Answer 18

vacant

junction

Question 19

What happens as DNA is pulled through the RuvABC complex?

Answer 19

RuvC scans for its preferred target sequence

Question 20

What do RuvA and RuvC directly target?

Answer 20

X junctions(specifically recognise 4 stranded Holliday junctions

Question 21

What stabilises the RuvABC complex on the Holliday junction?

Answer 21

RuvAB & RuvBC interact to stabilise the RuvABC complex

Question 22

What does RuvC introduce?

Answer 22

Paired cleavages across the junction

Question 23

In RuvABC what moves the junction?

Answer 23

RuvBC moves the junction to the preferred RuvC cut site

Question 24

In RuvABC what is the MAIN summarised function of RuvAB and RuvC?

Answer 24

RuvAB: co-ordination of branch migration
RuvC: resolution of Holliday junctions

Question 25

What are the two main solutions to fix a double stranded DNA break and what proteins are involved?

Answer 25

Dissolution: RecQ & Topo III
Resolution: RuvABC - dual cuts made at branch points

Question 26

How is RecA loaded onto DNA?

Answer 26

At a double stranded DNA break RecBCD acts as an exonuclease to expose ssDNA with a 3’ end. RecBCD loads RecA on the ssDNA and homologous pairing and strand exchange occur

Question 27

Synthesis dependent strand annealing (SDSA)

Answer 27

The third method of repairing double stranded DNA breaks

Question 28

What is the main difference about SDSA?

Answer 28

It does not involve the formation of a 4-stranded Holliday junction

Question 29

Describe SDSA

Answer 29

The 3’ strand that invades a homologous partner chromosome is used to prime DNA synthesis. The D loop is then unwound by a helicase allowing RecA to re-assemble and locate the other end of the original break with its newly synthesised partner.
Always results in non-crossovers

Question 30

Extended synthesis dependent strand annealing (ESDSA)

Answer 30

Repairs damage induced by ionising radiation and desiccation.

Question 31

What are the two main approaches to resolving double Holliday junctions in double strand DNA break repair?

Answer 31

Dissolution by RecQ-TopoIII (always gives non-crossovers)

Branch migration and resolution by RuvABC (crossovers or non-crossovers)