Lecture 9 : Holliday Junction (HJ) Dissolution and Resolution

Why must mitotic cells limit crossover (CO) events?

• Prevent LOH and allelic exchange between homologs

• Avoid sister chromatid exchange (SCE), chromosome missegregation, and genomic instability

What are the two main HR product classes?

• Non-crossover (NCO): sequence restored without reciprocal flanking

• Crossover (CO): reciprocal exchange of flanking sequences between chromatids

How are NCOs commonly produced?

Via synthesis-dependant strand annealing (SDSA): strand invasion, extension, and annealing without forming stable double Holliday junctions (dHJs)

What is synthesis-dependant strand annealing (SDSA)?

Rad51-mediated invasion and extension followed by D-loop dissociation; extended 3’ and re-anneals to the other resected end and is ligated → NCO

What proteins promote SDSA?

Rad51 and mediators for invasion; helicases (e.g. Srs2/FBH1) and anti-recombinases that dismantle D-loops to favour SDSA over dHJ formation

What is a Holliday junction?

A four-way DNA junction formed when two duplexes exchange strands during recombination; can be single or double HJ (dHJ) intermediates

What is HJ dissolution vs resolution - core difference?

• Dissolution: non-cutting disentangling of dHJs by convergent branch migration and decatenation → NCO

• Resolution: endonucleolytic cleavage of HJs by resolvases → can yield CO or NCO depending on cut orientation

Which complex mediates HJ dissolution in Humans?

BLM helicase with TOP3A (and RM11/2) - the BTR (Bloom-Topoisomerase-RMI) complex - performs convergent branch migration and decatenation to produce NCOs

What biochemical activities are essential for dissolution?

ATP-dependant helicase (BLM) for branch migration and type 1A topisomerase (Top3A) for strand passage/decatenation; accessory RMI proteins stabilise complex

Describe the stepwise mechanisms of dissolution by Sgs1/Top3 (yeast BTR)

Convergent branch migration of dHJ by Sgs1 (BLM ortholog) → formation of hemicatenane → Top3-mediated strand pasage (decatenation) resolves hemicatenane → NCO product

What are HJ resolvases in humans?

Gen1 (Yen1 ortholog) and a set of SLX-MUS complexes (SLX1-SLX4 with MUS81-EME1) that cleaves HJs

How do the two human resolution pathways differ mechanistically?

SLX–MUS acts as a coordinated nuclease complex that incises branch points (often targeting nicked/junction substrates) and processes diverse DNA structures; GEN1 performs symmetric 1‑nuclease cuts on fully formed HJs to yield ligatable ends.

How does resolution outcome depend on cut orientation?

Cleavage across opposite strands produces COs or NCOs depending on which strand pairs are cut; symmetric cuts can produce COs with exchanged flanking regions.

Why are multiple pathways (dissolution + resolvases) beneficial?

Redundancy ensures all HJs removed before mitosis; dissolution minimizes COs while resolvases clear persistent structures that escaped dissolution.

What is RuvABC (bacterial) — key biochemical properties?

RuvA tetrameric junction-binding protein + RuvB AAA+ ATPase motor (branch migration) + RuvC endonuclease that cleaves HJs; ATP-dependent branch migration followed by symmetric cleavage by RuvC.

How does Srs2 (yeast) influence joint molecules?

Srs2 is an anti-recombinase helicase that dismantles Rad51 filaments and D-loops, preventing formation/persistence of joint molecules and promoting SDSA/NCO outcomes.

What happens to cells lacking Sgs1/BLM or TOP3A activity?

Increased crossovers, elevated SCEs, chromosomal rearrangements, and genomic instability; cancer predisposition (e.g., Bloom syndrome with BLM loss).

Why does Yen1/GEN1 regulation differ between yeast and humans regarding nuclear localization?

Yeast Yen1 is cell-cycle phosphorylated to control nuclear import/activation timing; human GEN1 is constitutively nuclear during mitosis and gains access to chromatin when nuclear envelope breaks down, removing the need for phosphorylation-dependent import.

How are HJ-processing pathways spatially/temporally controlled through the cell cycle?

Dissolution (BTR) active in S/G2 to avoid COs; resolvases are restricted or inhibited during S phase and activated in late G2/M (via phosphorylation, complex assembly, or nuclear access) to clear remaining HJs before anaphase.

What are consequences of failed HJ removal before mitosis?

Chromosome missegregation, mitotic catastrophe, persistent SCEs, and genome instability leading to cell death or oncogenic transformation.