TLS (also known as FUS) is an RNA-binding protein that contributes the N-terminal half of fusion oncoproteins implicated in the development of human liposarcomas and leukemias. Here we report that male mice homozygous for an induced mutation in TLS are sterile with a marked increase in the number of unpaired and mispaired chromosomal axes in pre-meiotic spermatocytes. Nuclear extracts from TLS(-/-) testes lack an activity capable of promoting pairing between homologous DNA sequences in vitro, and TLS(-/-) mice and embryonic fibroblasts exhibit increased sensitivity to ionizing irradiation. These results are consistent with a role for TLS in homologous DNA pairing and recombination.

We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between S and S ␥ sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.The constant (C) 1 region of its heavy (H) chain determines the class of an Ig molecule. Mouse H chains are designated , ␦, ␥3, ␥1, ␥2b, ␥2a, ⑀, and ␣. Upon stimulation by antigen, expression of the early IgM class usually changes to that of another class (IgG3, IgG1, IgG2b, IgG2a, IgE, or IgA). At the DNA level, the process is mediated by recombination that generally involves DNA transfer between a switch (S) region within the intron 5Ј to C and another S region within the intron 5Ј to the particular C-gene segment that is to be expressed (1-5).S regions, several kilobase pairs in length, contain short, G-rich repetitive sequence elements, often arranged in tandem arrays. They may adopt secondary structures, such as stem loops, and many break points are located at such structures (6). In marked contrast to those observed in V(D)J rearrangement (7-11), the switch recombination break-and-rejoining points are imprecise. They lie scattered all over the S regions or, less often, even outside the S regions. Thus, class switch recombination is region-specific rather than site-specific. Rearrangements combining three S regions are occasionally detected (12), and, although switch recombination is usually intrachromosomal (13), intermolecular rearrangements have also been observed (14).Following transcriptional activation (3, 15), switch rearrangements generally result in deletion of the DNA sequences between the two breakpoints. The mechanism for most class switching events can be described by a loop-excision model (16). The exact structure of the loop intermediate is unknown; it may resemble a crossed (␣) or a stem loop (⍀) conformation. In that model, after loop formation, four DNA ends are generated by endonucleolytic cleavage of the two S regions to be recombined; these ends are possibly protected or even fixed by proteins. Eithe...

Recombination protein complex RC‐1, purified from calf thymus nuclear extracts, catalyzes cell‐free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure‐specific endonuclease co‐purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC‐1. N‐terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno‐cross‐reactivity of the RC‐1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC‐1‐mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC‐1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single-and doublestranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA doublestrand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/ TLS require Mg 2؉ and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.Faithful repair of DNA double-strand breaks (DSBs) 1 is of vital importance for maintenance of genomic integrity of cells. DSBs are generated by chemical damaging agents and ionizing radiation and are specifically induced during meiosis. Unrepaired or aberrantly repaired DSBs can lead to chromosomal rearrangements, eventually resulting in the formation of tumors or cell death. In mammalian cells, two major pathways for DSB repair are non-homologous end joining and homologous recombination.In Escherichia coli, RecA protein plays a crucial role in DSB repair by promoting homologous pairing and strand exchange between homologous DNAs (1, 2). Discovery of homologues of RecA in every eukaryote examined has underscored conservation of similar repair mechanisms throughout evolution (3, 4). The homologue of recA in Saccharomyces cerevisiae, the RAD51 gene, is a member of the RAD52 epistasis group required for genetic recombination and DSB repair (5). High expression of the mammalian RAD51 protein in meiotic and lymphoid tissues (6) and its localization in synaptonemal complexes early in meiosis (7-9), relocalization after treatment with DNA-damaging agents (7), and induction after stimulation of B-cells for class switch recombination (10) suggest a role for mammalian RAD51 in DNA recombination and repair. The finding that human and yeast Rad51 proteins promote ATP-dependent homologous pairing and strand exchange further support their central role in recombination and DSB repair (11-13).Although these studies indicate that the basic mechanism and key players of DSB repair are evolutionarily conserved, the process in higher eukaryotes is much more complex than in E. coli and involves a number of additional factors. In this context, evidence is accumulating for a possible role for the tumor suppressor proteins p53, BRCA1, and BRCA2 in DNA recombination and repair (4,14,15). This includes interaction of p53, BRCA1, and BRCA2 with RAD51; colocalization of BRCA1, BRCA2, and RAD51 on the axial elements of the synaptonemal complexes during meiosis; similarities i...

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the Cterminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for doublestranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffoldassociated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.The structural maintenance of chromosomes (SMC) 1 protein family, with members from lower and higher eukaryotes, may be divided into four subfamilies (SMC1 to SMC4) and two SMC-like protein subfamilies (SMC5 and SMC6) (for reviews see Refs. 1-7). Members of this family appear to function primarily as heterodimers and are implicated in a large range of activities that modulate chromosome structure and organization. Studies of yeast strains deficient in SMC1 and SMC2 show defects in the segregation of mitotic chromosomes (8 -10). A role in chromosome condensation was demonstrated in a cell-free chromosome condensation assay based on Xenopus laevis oocyte extracts (11), in which SMC2 and SMC4 subtypes were identified as essential components of the condensin protein complex (12). More recently, the Smc1p and Smc3p of Saccharomyces cerevisiae were shown to be essential for sister chromatid cohesion (13,14), and in Caenorhabditis elegans, two SMC protein homologs, MIX-1 and DPY-27, are involved in gene dosage compensation (15, 16). Finally, evidence for a role of SMC proteins in DNA recombination and repair has been supported by both genetic and biochemical studies. The Schizosaccharomyces pombe Rad18 gene and its S. cerevisiae homolog RHC18 are SMC-like and were found to act in an unusual postreplicative recombinational repair pathway (17), whereas mammalian SMC1 and SMC3 proteins were described as essential subunits of a recombinational repair protein complex (RC-1), isolated from calf thymus (18,19).SMC proteins display a very characteristic structure: two coiled-coil domains separate evolutionarily conserved head and tail domains, which contain an NTP binding motif (Walker A box) in the N terminus, and a DA box (Walker B) in the C terminus (20). The similarity of SMC proteins to motor proteins such as kinesin a...