PMID- 10601032
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  - Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in
      RAG1 critical for both cleavage steps of V(D)J recombination.
PG  - 3059-69
AB  - RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the
      antigen-binding domain of immunoglobulins and T-cell receptors, by introducing
      site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte
      development. These breaks are generated in two steps, nicking of one strand
      (hydrolysis), followed by hairpin formation (transesterification). The nature and
      location of the RAG active site(s) have remained unknown. Because acidic amino
      acids have a critical role in catalyzing DNA cleavage by nucleases and
      recombinases that require divalent metal ions as cofactors, we hypothesized that 
      acidic active site residues are likewise essential for RAG-mediated DNA cleavage.
      We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed
      mutagenesis, and examined >100 mutants using a combination of in vivo and in
      vitro analyses. No conserved acidic amino acids in RAG2 were critical for
      catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically
      inactive for both nicking and hairpin formation. These data argue that one active
      site in RAG1 performs both steps of the cleavage reaction. Amino acid
      substitution experiments that changed the metal ion specificity suggest that at
      least one of these three residues contacts the metal ion(s) directly. These data 
      suggest that RAG-mediated DNA cleavage involves coordination of divalent metal
      ion(s) by RAG1.
FAU - Landree, M A
AU  - Landree MA
AD  - Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas
      77030, USA.
FAU - Wibbenmeyer, J A
AU  - Wibbenmeyer JA
FAU - Roth, D B
AU  - Roth DB
LA  - eng
GR  - AI-07495/AI/NIAID NIH HHS/United States
GR  - T32 AI007495/AI/NIAID NIH HHS/United States
GR  - GM-08231/GM/NIGMS NIH HHS/United States
GR  - T32 GM008231/GM/NIGMS NIH HHS/United States
GR  - AI-36420/AI/NIAID NIH HHS/United States
GR  - R01 AI036420/AI/NIAID NIH HHS/United States
PT  - Comparative Study
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 (Recombinant Fusion Proteins)
RN  - 0 (V(D)J recombination activating protein 2)
RN  - 128559-51-3 (RAG-1 protein)
RN  - 42Z2K6ZL8P (Manganese)
RN  - 9007-49-2 (DNA)
RN  - EC 2.7.7.- (DNA Nucleotidyltransferases)
RN  - EC 2.7.7.- (VDJ Recombinases)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Amino Acid Sequence
MH  - Amino Acid Substitution
MH  - Animals
MH  - Binding Sites
MH  - CHO Cells
MH  - Catalysis
MH  - Catalytic Domain
MH  - Cricetinae
MH  - Cricetulus
MH  - Cysteine/chemistry
MH  - DNA/genetics/metabolism
MH  - DNA Nucleotidyltransferases/*metabolism
MH  - DNA-Binding Proteins
MH  - Gene Rearrangement/*physiology
MH  - Homeodomain Proteins/chemistry/genetics/*physiology
MH  - Humans
MH  - Lymphocytes/metabolism
MH  - Manganese/physiology
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Nuclear Proteins
MH  - Recombinant Fusion Proteins/physiology
MH  - Sequence Alignment
MH  - Sequence Homology, Amino Acid
MH  - Structure-Activity Relationship
MH  - Transfection
MH  - VDJ Recombinases
MH  - Vertebrates/genetics
PMC - PMC317185
EDAT- 1999/12/22 09:00
MHDA- 2000/02/19 09:00
CRDT- 1999/12/22 09:00
PHST- 1999/12/22 09:00 [pubmed]
PHST- 2000/02/19 09:00 [medline]
PHST- 1999/12/22 09:00 [entrez]
AID - 10.1101/gad.13.23.3059 [doi]
PST - ppublish
SO  - Genes Dev. 1999 Dec 1;13(23):3059-69. doi: 10.1101/gad.13.23.3059.