Visualizing Protein–DNA Interactions

Homologous recombination — synthesis-dependent strand annealing

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The mechanism

A double-strand break is repaired from the sister chromatid. The MRN complex senses the broken ends, and nucleases resect the 5′ strands, leaving 3′ single-stranded overhangs. RPA coats that single-stranded DNA at once and protects it.

BRCA2 then loads RAD51, which displaces RPA and polymerizes into a filament along the overhang. The filament finds the matching sequence in the sister chromatid and invades it; the 3′ end is extended using the sister as template, the new strand is released and anneals to the other end of the break, and the remaining gap is filled and sealed. This pathway is synthesis-dependent strand annealing.

1. 1 Double-strand break

   Both strands are cut at the same position

2. 2 End sensing

   MRN binds the broken ends

3. 3 Short-range resection

   MRN, with BRCA1, trims the 5′ ends

   • BRCA1 joins MRN at the break and favours resection
   • The 5′ end of each fragment is shortened, exposing short 3′ overhangs

4. 4 Long-range resection

   EXO1 extends the 3′ overhangs

   • EXO1 continues 5′→3′ from where MRN stopped
   • Each fragment now ends in a long 3′ single-stranded tail

5. 5 RPA coats the overhangs

   RPA protects both 3′ single-stranded tails

   • The resection machinery leaves the ends
   • Three RPA complexes coat each 3′ overhang side by side, removing secondary structure

6. 6 BRCA2 nucleates RAD51

   RAD51 replaces RPA at the junction of the right end

   • BRCA2 brings RAD51 to the single-strand–duplex junction
   • The RPA complex nearest the junction is displaced
   • Two RAD51 protomers take its place and bind each other

7. 7 RAD51 filament formation

   The filament grows towards the break, displacing RPA

   • RPA is displaced from the rest of the right overhang
   • Four more RAD51 protomers extend the filament, each bound to its neighbour
   • The RAD51 filament is ready to search for the matching sequence

8. 8 Engagement with the sister chromatid

   The filament aligns with the sister, which opens

   • BRCA2 leaves the assembled filament
   • The RAD51 filament also rests on the corresponding region of the sister chromatid
   • The sister duplex unwinds there; no base pair between the two molecules exists yet

9. 9 Strand invasion

   The 3′ overhang pairs with the complementary sister strand

   • The right 3′ overhang pairs with the sister strand it corresponds to
   • The other sister strand is left unpaired in the bubble, displaced
   • RAD51 is still on the strand that has just paired

10. 10 Synthesis on the sister chromatid

    The 3′ end is extended using the sister as template

    • The RAD51 filament disassembles, freeing the 3′ end
    • The sister unwinds ahead of the 3′ end
    • Pol δ extends the invading strand, copying the sister across the position of the break

11. 11 Displacement of the new strand

    The extended strand leaves the sister, which closes again

    • Pol δ leaves
    • The extended strand unpairs from the sister chromatid; it is still unpaired
    • The sister chromatid re-anneals, unchanged

12. 12 Annealing to the second end

    The new strand pairs with the other 3′ overhang

    • RPA leaves the left 3′ overhang
    • The newly synthesised strand anneals to it, bridging the break

13. 13 Gap filling and ligation

    The remaining gap is filled and the chromatid is intact

    • Pol δ extends the left 3′ end, copying the strand that now bridges the break
    • The remaining nicks are sealed
    • Both chromatids are intact; the repaired one carries two newly synthesised tracts

A schematic, explanatory model: it shows the order of events, not stoichiometry or kinetics.

How Molecular Motion represents it

A protein–DNA interaction here is more than an arrow between two shapes. The DNA has coordinates: an 80-base-pair chromatid with the break at position 40 and each overhang declared as a span. A protein declares a footprint, the number of nucleotides it covers and the form of DNA it binds, so RPA (form: single) can only sit on single-stranded DNA.

coat places several copies of a protein side by side on a span, and vacate removes one. The compiler keeps track of occupancy, so two proteins cannot be put on the same nucleotides: RAD51 can only take the place RPA has left. copies: 6 declares six RAD51 protomers, addressed as rad51#1 to rad51#6, each with its own state.

Protein–protein contacts within the filament use interfaces: each RAD51 has a protomer interface with a valence of two, and each bind between neighbours uses one. The strands themselves are part of the state too: resect, unwind, invade, extend and anneal change which nucleotides exist and which are paired, and the figure is drawn from that.

actors:
  - id: dna
    type: dna
    label: Broken chromatid
    compartment: nucleus
    nucleic: { length: 80 }
    sites:
      - id: break
        type: double-strand-break
        at: 40
      - id: left-overhang
        label: 3′ overhang (left end)
        span: [22, 40]
      - id: right-overhang
        label: 3′ overhang (right end)
        span: [40, 58]

  - id: rpa
    type: protein
    label: RPA
    color: "#e0a03c"
    compartment: nucleus
    copies: 6
    footprint: { length: 6, form: single }

  - id: rad51
    type: protein
    label: RAD51
    color: "#818cf8"
    compartment: nucleus
    copies: 6
    # `any` puts no form restriction on this footprint, so the filament may stay on the strand while
    # it pairs. It is not a statement about what RAD51 binds in general (RFC 0006 §7).
    footprint: { length: 3, form: any }
    interfaces:
      - { id: protomer, valence: 2 }

steps:
  - id: nucleation
    title: BRCA2 nucleates RAD51
    summary: RAD51 replaces RPA at the junction of the right end
    keyEvents:
      - BRCA2 brings RAD51 to the single-strand–duplex junction
      - The RPA complex nearest the junction is displaced
      - Two RAD51 protomers take its place and bind each other
    actions:
      - { type: vacate, actor: "rpa#3" }
      - { type: hide, actor: "rpa#3" }
      - type: coat
        actors: ["rad51#1", "rad51#2"]
        target: dna
        span: [52, 58]
        orientation: reverse
      - { type: bind, actor: "rad51#2", interface: protomer, target: "rad51#1", targetInterface: protomer }
      - { type: bind, actor: brca2, target: "rad51#1" }

That is part of the complete file. Showing it on a page takes a few lines:

import { parseMechanism } from '@molecular-motion/core/yaml';
import { MolecularMechanism } from '@molecular-motion/react';
import source from './homologous-recombination.yaml?raw';   // Vite; any way of reading the file as text works

export function Figure() {
  return <MolecularMechanism definition={parseMechanism(source)} controls />;
}

The language describes actors, sites and coordinates, and Actions lists every action used here. To change this mechanism and see the result, paste the file into the playground.

Related examples

• DNA repair: base excision repair and PARP1

  A single-strand break, detected and signalled by PARP1.

• Double Holliday junction

  The other outcome of strand invasion: a crossover.

• Cell signalling: cGAS–STING

  A protein that assembles on double-stranded DNA in the cytosol.

All examples · About Molecular Motion · Source on GitHub