PMID- 10601033
OWN - NLM
STAT- MEDLINE
DCOM- 20000111
LR  - 20190516
IS  - 0890-9369 (Print)
IS  - 0890-9369 (Linking)
VI  - 13
IP  - 23
DP  - 1999 Dec 1
TI  - Mutations of acidic residues in RAG1 define the active site of the V(D)J
      recombinase.
PG  - 3070-80
AB  - The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding
      recombination signal sequences (RSSs) and making a double-strand break between
      the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins,
      the bacterial transposases and retroviral integrases, cleavage by the RAG
      proteins requires a divalent metal ion but does not involve a covalent
      protein/DNA intermediate. In the transposase/integrase family, a triplet of
      acidic residues, commonly called a DDE motif, is often found to coordinate the
      metal ion used for catalysis. We show here that mutations in each of three acidic
      residues in RAG1 result in mutant derivatives that can bind the RSS but whose
      ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking
      and hairpin formation) is severely impaired. Because both chemical steps are
      affected by the same mutations, a single active site appears responsible for both
      reactions. Two independent lines of evidence demonstrate that at least two of
      these acidic residues are directly involved in coordinating a divalent metal ion:
      The substitution of Cys for Asp allows rescue of some catalytic function, whereas
      an alanine substitution is no longer subject to iron-induced hydroxyl radical
      cleavage. Our results support a model in which the RAG1 protein contains the
      active site of the V(D)J recombinase and are interpreted in light of predictions 
      about the structure of RAG1.
FAU - Kim, D R
AU  - Kim DR
AD  - Molecular Biology Department, Massachusetts General Hospital, Boston,
      Massachusetts 02114, USA.
FAU - Dai, Y
AU  - Dai Y
FAU - Mundy, C L
AU  - Mundy CL
FAU - Yang, W
AU  - Yang W
FAU - Oettinger, M A
AU  - Oettinger MA
LA  - eng
GR  - GM58026/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Genes Dev
JT  - Genes & development
JID - 8711660
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Homeodomain Proteins)
RN  - 0 (Nuclear Proteins)
RN  - 0 (RAG2 protein, human)
RN  - 0 (Rag2 protein, mouse)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (V(D)J recombination activating protein 2)
RN  - 128559-51-3 (RAG-1 protein)
RN  - 30KYC7MIAI (Aspartic Acid)
RN  - 3352-57-6 (Hydroxyl Radical)
RN  - 42Z2K6ZL8P (Manganese)
RN  - E1UOL152H7 (Iron)
RN  - EC 2.7.7.- (DNA Nucleotidyltransferases)
RN  - EC 2.7.7.- (VDJ Recombinases)
SB  - IM
MH  - Amino Acid Sequence
MH  - Amino Acid Substitution
MH  - Animals
MH  - Aspartic Acid/chemistry
MH  - Binding Sites
MH  - Catalytic Domain
MH  - DNA Nucleotidyltransferases/*chemistry/metabolism
MH  - DNA-Binding Proteins
MH  - Gene Rearrangement/*physiology
MH  - HeLa Cells
MH  - Homeodomain Proteins/*chemistry/genetics/metabolism
MH  - Humans
MH  - Hydroxyl Radical
MH  - Iron/pharmacology
MH  - Manganese/pharmacology
MH  - Mice
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Nuclear Proteins
MH  - Phenotype
MH  - Protein Binding
MH  - Protein Structure, Secondary
MH  - Protein Structure, Tertiary
MH  - Recombinant Fusion Proteins/physiology
MH  - Structure-Activity Relationship
MH  - VDJ Recombinases
PMC - PMC317176
EDAT- 1999/12/22 00:00
MHDA- 1999/12/22 00:01
CRDT- 1999/12/22 00:00
PHST- 1999/12/22 00:00 [pubmed]
PHST- 1999/12/22 00:01 [medline]
PHST- 1999/12/22 00:00 [entrez]
AID - 10.1101/gad.13.23.3070 [doi]
PST - ppublish
SO  - Genes Dev. 1999 Dec 1;13(23):3070-80. doi: 10.1101/gad.13.23.3070.