Human 75-kDa DNA-pairing Protein Is Identical to the Pro-oncoprotein TLS/FUS and Is Able to Promote D-loop Formation

Abstract

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...

The RNA-binding Protein Fused in Sarcoma (FUS) Functions Downstream of Poly(ADP-ribose) Polymerase (PARP) in Response to DNA Damage

“…Although other studies have implicated a role for FUS in maintaining resistance to genotoxic stress, possibly via participation in HR repair (34,40,41), the present results indicate that FUS functions are required for optimal DSB repair through both NHEJ and HR. FUS joins a growing list of RNA-processing factors including RBMX, PPM1G, THRAP3, hnRNPUL1 and -2, and NONO, that are recruited to the proximity of DSBs and whose activities are required for genotoxin resistance and DNA repair (51)(52)(53)(54).…”

“…Previous work has shown that FUS is capable of supporting D-loop formation, which suggests a potential role in HR (34). To test this we used the HEK-293 DR-GFP reporter cell line, which specifically measures repair through HR (46).…”

“…FUS Ϫ/Ϫ mice exhibit defects in B lymphocyte development and activation, male sterility, chromosomal instability, and radiosensitivity, phenotypes that are closely aligned with DSB repair defects (40,41). Cellular extracts from FUS Ϫ/Ϫ testes are unable to promote pairing between homologous DNA sequences in vitro (41), and FUS was shown to promote D-loop formation (34), an essential step in the repair of DSBs through the HR pathway (42). Thus, the available evidence suggests that FUS participates in HR repair, possibly through direct actions at DSBs.…”

“…2A) (31). FUS binds RNA (32,33) and ssDNA and dsDNA (33)(34)(35) and has been shown to shuttle between the nucleus and cytoplasm (33). In addition to its participation in transcription, FUS has proposed physiological activities involving microRNA processing, splicing, and mRNA transport and maturation (36,37).…”

Multiple functional domains of the oncoproteins Spi-1/PU.1 and TLS are involved in their opposite splicing effects in erythroleukemic cells

“…These data agree with a role of TLS in transcriptional regulation. TLS has been biochemically characterized as hPOMp75, a protein involved in homologous DNA pairing and recombination (Baechtold et al, 1999;Bertrand et al, 1999). This activity fits with the increased sensitivity to ionizing radiation due to chromosome pairing defects during meiosis in the TLS-deficient mice suggesting that TLS could participate to the maintenance of genomic integrity (Kuroda et al, 2000).…”