Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination.
Mol Cell Biol
; 18(1): 655-63, 1998 Jan.
Article
in En
| MEDLINE
| ID: mdl-9418911
ABSTRACT
We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Recombination, Genetic
/
Receptors, Antigen, T-Cell
/
Genes, RAG-1
/
Homeodomain Proteins
/
DNA Footprinting
/
DNA-Binding Proteins
Limits:
Animals
/
Humans
Language:
En
Journal:
Mol Cell Biol
Year:
1998
Type:
Article
Affiliation country:
Japan