During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 create

During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 create DNA increase strand breaks within a combined complex (PC) including two complementary recombination sign sequences (RSSs), the 23RSS and 12RSS, which vary in the space of the spacer separating heptamer and nonamer elements. U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site 371935-74-9 manufacture and contributes to the 12/23 rule. INTRODUCTION V(D)J recombination assembles and diversifies the variable region of the antigen receptor genes of B and T lymphocytes. The process requires the recombination activating gene proteins RAG1 and RAG2 (together referred to as RAG), which interact with one another and perform DNA binding and cleavage functions to initiate V(D)J recombination (1C3). RAG specifically binds recombination signal sequences (RSSs) that flank the V (variable), D (diversity) and J (joining) coding gene segments that are the substrates of the reaction. RSSs consist of well conserved heptamer and nonamer elements separated by a less well conserved spacer region of 12 bp or 23 bp (referred to as the 12RSS or 23RSS, respectively) (Figure 1B). Figure 1. V(D)J recombination and the FRET assay for DNA bending. (A) Steps during V(D)J recombination. The complex containing RAG1, RAG2 and HMGB1/2 is represented as the green shape, V and J coding segments as rectangles as well as the 12RSS and 23RSS as reddish colored and blue … The first step in V(D)J recombination is usually binding of RAG, probably together with high mobility group box protein (HMGB) 1 or HMGB2, to either a 12RSS or a 23RSS to form the 12 signal complex (12SC) or the 23SC, respectively (Physique 1A). The SC is usually then thought to capture a protein-free partner RSS (4,5) in a process known as synapsis to form the paired complex (PC). DNA cleavage takes place by a two-step nick-hairpin mechanism (Physique 1B) at each RSS, resulting in two double-strand breaks that individual the coding ends from the signal ends. Although the nicking step can occur in the SC before synapsis, hairpin formation is largely restricted to the PC. The DNA ends are subsequently processed 371935-74-9 manufacture and joined by factors of the nonhomologous end joining repair pathway (6,7). The generation of double strand breaks and the overall recombination reaction occur much more efficiently with a 12/23 RSS pair than with 12/12 or 23/23 RSS pairs, a preference known as the 12/23 rule. The 12/23 rule appears to be imposed at both the synapsis and hairpin formation actions of the reaction (1,2,8). HMGB1 or HMGB2, nonspecific DNA-binding proteins 371935-74-9 manufacture that are capable of stabilizing/inducing DNA bends (9), are vital in vitro for efficient formation of the PC and hence for hairpin formation. They are also important for formation of the 23SC but less so for formation of the 12SC (1,8). RAGCRSS interactions have been studied in detail with footprinting, interference and photo-crosslinking methodologies [reviewed in (8)], revealing extensive interactions with the nonamer and nonamer-proximal spacer by RAG1 and in and 371935-74-9 manufacture around the heptamer, probably by both RAG1 and RAG2. RAG interactions with the phosphate backbone are detected primarily on one side of the DNA helix and are comparable for the 12RSS and the 23RSS. HMGB1/2-RSS contacts, although less well defined, have been proposed to occur at the spacer/nonamer border, Smcb near the site of cleavage and within the 23RSS spacer (8,10,11). Consistent with the stable incorporation of a DNA bending protein into RAGCRSS complexes, several previous studies have provided evidence for DNA bends in RAGCRSS complexes, although there is usually little information about nature and magnitude of bends in the PC (12C16) (see Discussion). Minimal core regions required for DNA cleavage and recombination activity have been defined for murine RAG1 (amino acids 384C1008) and RAG2 (amino acids 1C387). The RAG1 core (RAG1c) contains heptamer and nonamer recognition domains as well as the active site for DNA cleavage. RAG2c appears to function as a cofactor 371935-74-9 manufacture for RAG1, improving DNA.