Nucleosome structure completely inhibits in vitro cleavage by the V(D)J recombinase.
Lineage specificity and temporal ordering of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In this report, we investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. We found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. We suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure.
Johns Hopkins University School of Medicine, Department of Medicine, Department of Molecular Biology and Genetics, 1068 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205, USA.
A
A
Golding
S
S
Chandler
E
E
Ballestar
A P
AP
Wolffe
M S
MS
Schlissel
D000107:Acetylation; D000818:Animals; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004912:Erythrocytes; D015321:Gene Rearrangement / Q000235:genetics; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008274:Magnesium; D015394:Molecular Structure; D009687:Nuclear Proteins; D009707:Nucleosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D045567:VDJ Recombinases
D002843:Chromatin; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D006657:Histones; D018398:Homeodomain Proteins; D007145:Immunoglobulin kappa-Chains; D009687:Nuclear Proteins; D009707:Nucleosomes; C499311:RAG2 protein, human; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D014357:Trypsin; D008274:Magnesium