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 re combination signal sequences (RSSs) within chromatin to in vitro cleavage b y 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 ini tiate cleavage on positioned nucleosomes containing RSS substrates. We foun d that nicking and double-strand DNA cleavage of RSSs positioned on the fac e of an unmodified nucleosome are entirely inhibited. This inhibition was i ndependent of translational position or rotational phase and could not be o vercome either by addition of the DNA-bending protein HMG-1 or by the use o f hyperacetylated histones, me suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potenti al RSS targets, and that actively rearranging gene segments might be packag ed in a modified or disrupted nucleosome structure.