h Interacts with the lmmunoglobulin V-J Recombination Sequences

of Microbiology and Immunology University of California Berkeley, California 94720 Summary DNA-nuclear protein interactions were studied with synthetic recombination signal sequences (RSSs) for immunoglobulin V-J joining. With a gel retardation as- say, a DNA-binding protein that specifically interacts with RSSs was detected in nuclear extracts from a pre- B cell line, 3889. This protein was found in all the recombination-competent pre-B cell lines tested in this study, but not in myeloma, mature T cell, mono- cyte, or fibroblast cell lines. DNA footprint analysis with dimethyl sulfate demonstrated that the 7-mer re- gion of the RSS was strongly protected when com- plexed with the binding protein. Furthermore, a single base substitution in the Fmer region totally abolished the binding. The molecular mechanism of V-J joining is discussed in the context of the RSS-binding protein. Introduction Antigen receptor genes are generated by site-specific DNA recombination both in T cells and 6 cells during the differentiation of lymphocytes (reviewed by Kronenberg et al., 1986; Tonegawa, 1983). On the germ-line chromo- some, variable region genes for the antigen receptor are split into variable (V), diversity (D), and joining (J) seg- ments. These gene segments are brought together by a site-specific DNA recombination process known as V-(D)- J joining. This DNA rearrangement plays a key role not only in the activation of the antigen receptor genes, but also in the diversification of the gene sequences. To study the molecular mechanism of V-(D)-J joining, three different approaches have been taken. One ap- proach is the structural study of the recombination region by DNA cloning and sequencing. From these analyses it has been revealed that both immunoglobulin (lg) and T cell receptor (TCR) genes contain two highly conserved sequences adjacent to the recombination sites; they are a palindromic 7-mer, CACTGTG, and a T-rich 9-mer, GGTTTTTGT, separated by a spacer of constant length (Sakano et al., 1979, 1980, 1981; Max et al., 1979; Early et al., 1980; Davis, 1985). The second approach is to in- troduce artificial recombination substrates with appropri- ate selection markers into recombination-competent pre-B cells (Lewis et al., 1984, 1985; Blackwell and Alt, 1984; Yancopoulos et al., 1986; Hesse et al., 1987). Alt and Balti- more and their colleagues have demonstrated that V-(D)-J joining can take place on exogenous lg or TCR genes in- troduced into the host chromosomal DNA with a retroviral vector or by DNA transfection. The third approach is to characterize the enzymatic machinery responsible for V-(D)-J joining. We assume that at least three activities are involved in the recombinase complex for V-(D)-J joining (Hope et al., 1986): a DNA-binding activity, an endonu- cleolytic activity, and a ligase activity. The DNA-binding activity would recognize the recombination sites and bring the two substrates together into the proper orientation; the endonucleolytic activity would cleave the germ-line seg- ments at the recombination sites; and finally, the ligase activity would join the two cleaved DNA substrates cova- lently to complete the joining reaction. Endonucleases presumably involved in the DNA-cutting activity of recombinase have been previously described (Desiderio and Baltimore, 1984; Kataoka et al., Hope 1986). However, little is known about other activities, and as yet the identification of a whole recombinase com- plex has not been reported. In the present study we have attempted to identify a DNA-binding protein(s) that interacts specifically with recombination signal sequences (RSSs). As a source of the protein, we have used nuclear extracts from an immature pre-B cell line, 3889, kindly provided by Dr. F. W. Alt. In this cell line lg gene rearrangement actively takes place during in vitro culture (Alt et al., 1981; Black- well and Alt, 1984). We synthesized DNA substrates con- taining the Fmer and 9-mer separated by either a 12 bp or a 23 bp spacer. Using the synthetic recombination sub- strates, we first verified that two sets of RSSs are sufficient to cause the V-J-type joining provided the 12/23 bp spacer rule (Sakano et al., 1980; Early et al., 1980) is satisfied. We then searched for a specific DNA-binding protein(s) in the pre-B cell nuclear extracts by using the synthesized RSS probes. With a gel retardation assay (Singh et al., 1986; Schneider et al., 1986), we have identified a nuclear pro- tein that can specifically interact, particularly in the Fmer region, with the RSS probe containing the 12 bp spacer. In this report we characterize this RSS-binding activity, which may be one of the DNA-binding components as- sociated with V-(D)-J joining. Results Synthetic Substrates Containing the Fmer and 9-mer Can Cause V-J-Type Recombination Two double-stranded oligonucleotides containing the Fmer and 9-mer were chemically synthesized (Figure 1A). One contained a 12 bp spacer separating the recombina- tion signals (12 bp-RSS), and the other contained a 23 bp spacer (23 bp-RSS). To study whether these two RSSs are sufficient substrates to mediate V-J-type recombina- tion, we constructed a plasmid, pKOdel-1. It contains a ne- omycin resistance gene (near) (Beck et al., 1982) flanked by two sets of RSSs, and a thymidine kinase gene (tk) (McKnight, 1980) further downstream (Figure 1B). Expres- sion of these genes is mediated by a promotor complex consisting of the SV40 and t/r gene promoters (McKnight, 1980; Southern and Berg, 1982).