Abstract
The immunoglobulin heavy chain switch regions contain multiple runs of guanines on the top (nontemplate) DNA strand. Here we show that LR1, a B cell-specific, duplex DNA binding factor, binds tightly and specifically to synthetic oligonucleotides containing G-G base pairs (KD </= 0.25 nM). LR1 also binds to single-stranded G-rich sequences (KD approximately 10 nM). The two subunits of LR1, nucleolin and hnRNP D, bind with high affinity to G4 DNA (KD = 0.4 and 0.5 nM, respectively). LR1 therefore contains two independent G4 DNA binding domains. We propose that LR1 binds with G-G-paired structures that form during the transcription of the S regions that is prerequisite to recombination in vivo. Interactions of donor and acceptor S regions with subunits of the LR1 could then juxtapose the switch regions for recombination.
Highlights
Immunoglobulin class switch recombination is a regulated recombination event that joins a rearranged and expressed heavy chain variable region (VDJ) to a new downstream constant (C)1 region, deleting the DNA between
To test the possibility that LR1 might recognize G4 DNA formed by S region sequences, we carried out gel mobility shift experiments to assay binding of highly purified protein to 5Ј end-labeled G4 DNA formed from the P oligonucleotide
We have shown that the B cell-specific factor, LR1, binds to G-rich S region sequences as Watson-Crick duplexes and as G-G-paired structures stabilized by Hoogsteen pairing
Summary
C, constant; hnRNP, human ribonucleoprotein; RBD, RNA binding domain. tion of the S regions in the heavy chain locus reflects their functional importance in switch recombination: there is an S region upstream of each C region except C␦, and use of the C␦ region is governed by RNA processing, not DNA recombination. The fact that G-rich DNA occurs at very specific regions of the genome suggests that G-G pairing may be important to specific cellular functions Consistent with this hypothesis, a number of proteins have been described that bind to, cleave, or promote formation of G-G-paired DNAs (10 –17), including some that interact with telomeric sequences (18 –21). RNA binding domains (RBDs, called RNA recognition motifs, or RRMs) and Arg-Gly-Gly repeats (RGGs) These structural motifs are commonly found in proteins that interact with RNA or single-stranded DNA Binding to G4 DNA and single-stranded DNA was carried out in 15-l reactions containing 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 100 g/ml bovine serum albumin, 1 fmol of 32P-labeled DNA for 30 min at 37 °C, and the complexes were resolved by gel electrophoresis on 6% polyacrylamide, 45 mM Tris-borate-EDTA gels at 4 °C.
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