Nucleolin is a very abundant eukaryotic protein that localizes to the nucleolus, where the rDNA undergoes transcription, replication, and recombination and where rRNA processing occurs. The top (non-template) strand of the rDNA is very guanine-rich and has considerable potential to form structures stabilized by G-G pairing. We have assayed binding of endogenous and recombinant nucleolin to synthetic oligonucleotides in which G-rich regions have formed intermolecular G-G pairs to produce either two-stranded G2 or fourstranded G4 DNA. We report that nucleolin binds G-Gpaired DNA with very high affinity; the dissociation constant for interaction with G4 DNA is K D ‫؍‬ 1 nM. Two separate domains of nucleolin can interact with G-Gpaired DNA, the four RNA binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains bind G4 DNA with high specificity and recognize G4 DNA structure independent of sequence context. The high affinity of the nucleolin/G4 DNA interaction identifies G-Gpaired structures as natural binding targets of nucleolin in the nucleolus. The ability of two independent domains of nucleolin to bind G-G-paired structures suggests that nucleolin can function as an architectural factor in rDNA transcription, replication, or recombination.Transcription and processing of rRNA occur within a specialized subnuclear compartment, the nucleolus. In cells that are actively transcribing the rDNA, nucleoli appear to be composed of three compartments: the fibrillar center, which contains DNA that is not being transcribed; the dense fibrillar component, where rDNA transcription occurs; and the peripheral granular component, where pre-rRNA processing and pre-ribo-

In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.

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LR1 is a B cell-specific, sequence-specific DNA binding activity that regulates transcription in activated B cells. LR1 also binds Ig heavy chain switch region sequences and may function in class switch recombination. LR1 contains two polypeptides, of 106 kDa and 45 kDa, and here we report that the 106-kDa component of LR1 is nucleolin. This identification, initially made by microsequence analysis, was verified by showing that (i) LR1-DNA binding activity increased in B cells transfected with a nucleolin cDNA expression construct; (ii) LR1-DNA binding activity was recognized by antibodies raised against recombinant human nucleolin; and (iii) in B cells transfected with epitope-tagged nucleolin expression constructs, the LR1-DNA complex was recognized by the anti-tag antibody. Nucleolin is an abundant nucleolar protein which is believed to play a role in rDNA transcription or organization, or rRNA processing. Homology between nucleolin and histone H1 suggests that nucleolin may alter DNA organization in response to cell cycle controls, and the nucleolin component of LR1 may therefore function to organize switch regions before, during, or after switch recombination. The demonstration that nucleolin is a component of a B cell-specific complex that binds switch region sequences suggests that the G-rich switch regions may have evolved from rDNA.LR1 is a B cell-specific, sequence-specific DNA binding activity. It was first identified as a factor that specifically recognizes Ig switch region sequences and is induced in primary B cells activated to carry out class switch recombination (1, 2). LR1 has also been shown to regulate transcription of two genes that function in B cell transformation, c-myc (3) and the Epstein-Barr virus EBNA-1 gene (4).In Ig switch recombination, one constant region is literally switched for another by joining a rearranged and expressed variable region to a downstream constant region, deleting a long region of intervening DNA. Switching involves repetitive, G-rich regions of DNA, called switch regions (S regions), that are found upstream of the constant regions that undergo switch recombination (5-7). A specific S region is targeted for recombination by induction of noncoding transcripts from a promoter upstream of that S region (8-14), and recombination depends on both transcription and splicing of these switch transcript (15). Since S region transcription is prerequisite to recombination, it is possible that LR1 binding to sites in the S regions might potentiate S region transcription and thereby activate recombination. In vitro DNA binding studies have shown that the LR1-DNA binding consensus, GGNC-NAG(G͞C)CTG(G͞A), is loose, and LR1 may bind multiple sites in each of the G-rich S regions. This suggests that another possible function for LR1 could be to organize S region DNA before, during, or after recombination.To understand the function of LR1, we have purified and characterized the activity. LR1 is a complex of two polypeptides, of 106 kDa and 45 kDa (2, 16). In this communicati...