Lecture 6 DNA Recombi 2 Resolution Flashcards
Rec-Q like helicases
RecQ helicases represent a highly conserved family required for the maintenance of genome integrity. Rec Q helicases function as the interface between DNA replication and recombination to repair damaged replication forks.
The family is defined by a highly conserved helicase domain that comprises of ~400 amino acids and includes 7 sequence motifs (l-Vll) including an ATP binding site that is characteristic of a wide variety of helicases that function to unwind DNA strands. The RQC domain is unique to RecQ family helicases and consists of a zinc binding domain and a winged helix (a type of helix-turn-helix) involved in replication fork recognition.
The HRDC domain is found in a number of nucleases that digest RNA and is involved in DNA binding.
RecQ aids DNA replication
Disrupts secondary structures like hairpins to allow smooth replisome ( i.e. DNA polymerase lll replication complex) progression. Unwind ENA with 3’5’ polarity, catalyze HJ branch migration fork regression and unwinding D loops, triple helices and quadruplex DNA
Coordinated action of RecQ helicase and Topo lll form a ‘dissolvasome’ complex critical for untangling intertwined DNA structures that can arise during DNA replication and repair. Topoisomerases relieve torsional stress arising from DNA supercoiling by temporarily breaking DNA backbone in one (type 1) or both (type ll e.g. DNA gyrase) strands and then passing intact DNA through the opening before resealing the break. Topolll is a type 1 topoisomerase.
Double HJ formed by DSBR can be dissolved by RecQ-Topolll. The 2 HJ are migrated towards each other to form a hemicatenane. The molecules are decatenated (untangled) by Topolll. Always leads to non-crossover products - promoting genome stability
Holliday junction resolution by dual strand scission
4 stranded HJ can branch migrate over long dist as the 2 paired DNA molecules are homologous.
Recombined duplexes may not be identical - some mismatches are tolerated.
The unfolded ‘open’ junction (the x) is optimal for branch migration. Resolution occurs by nicking an opposing pair of strands at the point of crossover.
Outcome non-crossover aka patch or non-recombinant
Or crossover aka splice or recombinant
Products of resolution by symmetrical dual strand cleavage at the branch point are nicked duplexes. Which can be resealed by DNA ligase.
Holliday junction resolution by RuvABC
In Gram-neg bacteria such as E. Coli the branch migration and resolution steps are combined and carried out by RuvABC proteins. RuvAB and ruvC genes are expressed from diff promoters. RuvAB perform branch migration while ruvC is an endonuclease that resolves HJ. Mutants of Ruv A B and C are sensitive to DNA damaging agents (e.g. UV hence resistance to UV = Ruv) and show a slight defect in homologous recombination. All 3 Ruv mutants show same phenotype (I e. Gene products work together)
RuvA
RuvA forms a tetramet with two channels running across the surface that assembles on HJ DNA. Specificity is achieved by two neg-charged residues from each monomer forming a protruding pin structure that blocks access of linear dsDNA. Changing these to basic residues produces a protein that readily binds to DNA duplexes. Two tetramers can bind to sandwich the junction.
RuvB
A hexamer of RuvB threads onto dsDNA through the centre of the protein rings (like beads on a strong) ATP hydrolysis drives the pump, which translocates along dsDNA. Unlike most other helicases it doesn’t unwind the DNA strands. Ruv A acts as a specificity factor, targeting RuvB rings to the junction. Two RuvB monomers target each tetramer block and then the full hexamers can be assembled. Together they promote movement or branch migration of HJ to extend heteroduplexes
Ruv C
HJ is resolved by the dimeric ruvC endonuclease. The pair of active sites mediate dual incisions on opposite strands at the junction branch point. Hydrolysis reaction requires magnesium bound at the active site, which consists of 4 neg charged residues. efficient cleavage requires a 4bp target (5’A/T TT↓G/C-3) the point of strand crossover has to move (branch migrate) to the target sequence before cleavage can occur
RuvABC resolvasome complex
Coordinated branch migration (RuvAB) and resolution (RuvC) of HJ. RuvA targets junction and promotes assembly of the RuvB rings. Ruv C fits on vacant side of the junction. As the DNA is pulled through the complex, Ruv C scans for it’s preferred target sequences. Some conformational change probably occurs prior to resolution and this may pop off RuvA before the junction is eliminated and the proteins are released.
RuvA and RuvC directly target X junctions (they can recognise 4 strand HJ)
Ruv AB and RuvBC interact and stabilise the RuvABC complex on HJ
RuvC introduces paired ravages across junction (resolution)
RuvAB move the junction to preferred RuvC cut sites
RuvC functions as part of complex with RuvAB
Resolution of double HJ in dsDNA break repairs
At dsDNA break RecBCD act as exonuclease to expose ssDNA sections with a 3’ end. RecBCD loads RecA onto this ssDNA and homologous pairing and strand exchange occur with an undamaged chromosome (3’ ends can prime DNA synthesis)
2 HJ are ultimately formed to bridge broken ends of the break
Double HJ can be eliminated by dissolution using RecQ and Topolll. Alternatively RuvABC branch migrate the 2 HJ and Ruv C makes dual cuts at branch point of each. Resolution of both junction by 2 vertical or 2 horizontal cuts will give non-crossover. One vertical and one horizontal cut gives a crossover.
Synthesis dependent strand annealing SDSA
DsDNA breaks can be repaired by another homology dependent process called synthesis dependent strand annealing. 4strand HJ not formed instead a 3 stranded D loop forms. The 3’ strand that invades a homologous partner chromosome is used to prime DNA synthesis. This D loop is then unwound by a helicase e.g. recQ allowing RecA to reassemble and locate the other end of the original breakwith it’s newly synthesised partner. SDSA always results in non crossovers.
Key points
RecQ helicase helps to eliminate DNA secondary structures that may interfere with replication
2 approaches to solving HJ in dsDNA break repair:
1) dissolution by recQ - Topolll (non-crossover)
2) branch migration and resolution by RuvABC (crossover or non-crossover)
SDSA gives only non-crossover
