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TitleCracking the DNA Code for V(D)J Recombination.
Journal, issue, pagesMol Cell, Vol. 70, Issue 2, Page 358-370.e4, Year 2018
Publish dateApr 19, 2018
AuthorsMin-Sung Kim / Watchalee Chuenchor / Xuemin Chen / Yanxiang Cui / Xing Zhang / Z Hong Zhou / Martin Gellert / Wei Yang /
PubMed AbstractTo initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have ...To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.
External linksMol Cell / PubMed:29628308 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution2.75 - 4.15 Å
Structure data

7470

6cg0

EMDB-7470: RAG1/2 HFC complex
PDB-6cg0: Cryo-EM structure of mouse RAG1/2 HFC complex (3.17 A)
Method: EM (single particle) / Resolution: 3.17 Å

7480

6cij

EMDB-7480, PDB-6cij:
Cryo-EM structure of mouse RAG1/2 HFC complex containing partial HMGB1 linker(3.9 A)
Method: EM (single particle) / Resolution: 3.9 Å

PDB-5zdz:
Hairpin Forming Complex, RAG1/2-Nicked 12RSS/23RSS complex in Ca2+
Method: X-RAY DIFFRACTION / Resolution: 2.8 Å

PDB-5ze0:
Hairpin Forming Complex, RAG1/2-Nicked(with Dideoxy) 12RSS/23RSS complex in Mg2+
Method: X-RAY DIFFRACTION / Resolution: 2.75 Å

PDB-5ze1:
Hairpin Forming Complex, RAG1/2-Nicked 12RSS/23RSS complex in 2mM Mn2+ for 10 min at 4'C
Method: X-RAY DIFFRACTION / Resolution: 3.0 Å

PDB-5ze2:
Hairpin Complex, RAG1/2-hairpin 12RSS/23RSS complex in 5mM Mn2+ for 2 min at 4'C
Method: X-RAY DIFFRACTION / Resolution: 3.3 Å

PDB-6cik:
Pre-Reaction Complex, RAG1(E962Q)/2-intact/nicked 12/23RSS complex in Mn2+
Method: X-RAY DIFFRACTION / Resolution: 3.15 Å

PDB-6cil:
PRE-REACTION COMPLEX, RAG1(E962Q)/2-INTACT/INTACT 12/23RSS COMPLEX IN MN2+
Method: X-RAY DIFFRACTION / Resolution: 4.15 Å

PDB-6cim:
Pre-Reaction Complex, RAG1(E962Q)/2-nicked/intact 12/23RSS complex in Mn2+
Method: X-RAY DIFFRACTION / Resolution: 3.6 Å

Chemicals

ChemComp-CA:
Unknown entry

ChemComp-ZN:
Unknown entry

ChemComp-EDO:
1,2-ETHANEDIOL

ChemComp-K:
Unknown entry

ChemComp-HOH:
WATER

ChemComp-MG:
Unknown entry

ChemComp-MN:
Unknown entry

Source
  • mus musculus (house mouse)
  • homo sapiens (human)
  • escherichia coli k-12 (bacteria)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN / V(D)J recombination / RAG1-2-12RSS-23RSS complex / Hairpin forming complex / Hairpin complex / RECOMBINATION / RAG1/2 / RSS / Immunity / RECOMBINATION/DNA / RECOMBINATION-DNA complex / VDJ recombination

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