Processing of DNA for nonhomologous end-joining by cell-free extract

Budman, Joe; Chu, Gilbert

doi:10.1038/sj.emboj.7600563

Cited by 119 publications

(118 citation statements)

References 46 publications

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“…Although DNAs with cohesive ends have been used as substrates for in vitro NHEJ assays, such assays do not necessarily entail the action of the nuclease or polymerase characteristic of NHEJ (3,4,32,65), and alternative substrates have been devised (5,30,42). We generated a 5.7-kb DNA substrate with incompatible protruding 3Ј ends (Fig.…”

Section: Resultsmentioning

confidence: 99%

The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining

Shamanna

Hoque

Lewis-Antes

et al. 2011

Molecular and Cellular Biology

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Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased ␥-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process.Nuclear factor 90 (NF90) and nuclear factor 45 (NF45) form a heterodimeric core complex (18) that is abundant in many cells and tissues (36,49,55,66). NF90 is a product of the interleukin enhancer-binding factor 3 gene, ILF3, and is also known as DRBP76, NFAR1, and TCP80. NF45, or ILF2, is encoded by the ILF2 gene.NF90 and NF45 interact with numerous proteins and RNAs, generating higher-order complexes that participate in biological processes, including transcription, RNA transport, mRNA stability, and translation (2, 51). NF90 and NF45 were initially purified as DNA binding proteins (8), however, and evidence for their involvement in DNA metabolism is accumulating.

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Section: Resultsmentioning

confidence: 99%

The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining

Shamanna

Hoque

Lewis-Antes

et al. 2011

Molecular and Cellular Biology

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“…Studies by the Chu lab (151) with fractionated cell extracts have shown that DNA ligase IV/XRCC4 is required for processing of DNA molecules with incompatible ends. This implies that DNA ligase IV/XRCC4 acts prior to and is necessary for the subsequent recruitment of end-processing activities, providing an explanation as to why DNA molecules with cohesive ligatable ends are predominantly joined without processing (152). Although Ku is required for the recruitment of DNA ligase IV/XRCC4 to double-strand breaks, there are contradictory reports as to the role DNA PK cs in this recruitment (153,154).…”

Section: Dna Ligase IVmentioning

confidence: 97%

Eukaryotic DNA Ligases: Structural and Functional Insights

Ellenberger

Tomkinson

2008

Annu. Rev. Biochem.

291

338

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DNA ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA ligases. Members of the DNA ligase I and IV families are found in all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.

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“…NHEJ has been reconstituted using purified components from mammals (52) and yeast (16). More-extensive studies, to date limited to mammalian cells and Xenopus laevis, have examined DNA end joining in cell and nuclear extracts (47), where Ku-dependent NHEJ (4,12,14,17,42,46), as well as the other forms of end joining observed in vivo (14,17,32,46,50,60,67,73), can be seen.…”

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Ku Heterodimer-Independent End Joining inTrypanosoma bruceiCell Extracts Relies upon Sequence Microhomology

et al. 2007

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DNA double-strand breaks (DSBs) are repaired primarily by two distinct pathways: homologous recombination and nonhomologous end joining (NHEJ). NHEJ has been found in all eukaryotes examined to date and has been described recently for some bacterial species, illustrating its ancestry. Trypanosoma brucei is a divergent eukaryotic protist that evades host immunity by antigenic variation, a process in which homologous recombination plays a crucial function. While homologous recombination has been examined in some detail in T. brucei, little work has been done to examine what other DSB repair pathways the parasite utilizes. Here we show that T. brucei cell extracts support the end joining of linear DNA molecules. These reactions are independent of the Ku heterodimer, indicating that they are distinct from NHEJ, and are guided by sequence microhomology. We also demonstrate bioinformatically that T. brucei, in common with other kinetoplastids, does not encode recognizable homologues of DNA ligase IV or XRCC4, suggesting that NHEJ is either absent or mechanistically diverged in these pathogens.Two principal mechanisms have been described for the repair of DNA double-strand breaks (DSBs). In one pathway, termed homologous recombination, the DSB end(s) invades an intact homologous duplex, allowing DNA resynthesis and religation. Homologous recombination appears to be universal, since its core enzyme is conserved in the three kingdoms of life (31) and in viruses: RecA in bacteria, Rad51 in eukaryotes, RadA in archaea, and UvsX in phageT4 (34). The other pathway, termed nonhomologous end joining (NHEJ), rejoins a DSB directly, without relying on sequence homology. NHEJ is also widely conserved, since the factors involved have been described in all eukaryotes to date (22, 41) and a related process, catalyzed by conserved enzymes, is present in some bacterial lineages (10).Eukaryotic NHEJ is a multistep reaction catalyzed by a core set of conserved proteins, comprising the KU70-KU80 heterodimer (Ku) and a complex of DNA ligase IV (Lig IV; Dnl4 in Saccharomyces cerevisiae) and XRCC4 (Lif1 in yeast). Ku binds as a ring to DSB ends, where it can translocate along the duplex (76), and appears to bridge the DNA termini and recruit the other factors for end processing and ligation. DNA Lig IV appears to have evolved specifically for NHEJ, since it interacts with Ku (58) and does not complement the functions of other cellular ligases, which do not function in NHEJ (54, 80). Monomeric DNA Lig IV forms a stable, symmetrical complex with a dimer of XRCC4 (29, 69), which appears to stabilize and activate the ligase and to target it to DSBs. Beyond this core NHEJ machinery, a number of other proteins contribute to the reaction. NEJ1/Lif2 in yeast (33, 74) and XLF/Cernunnos in vertebrates (1, 11) interact with Lig IV-XRCC4 and share sequence similarities with XRCC4 (13), perhaps constituting a protein family whose members are distributed unevenly among eukaryotes (13, 64). In mammals, Ku bound to DNA constitutes two subunits of DNA-depen...

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Processing of DNA for nonhomologous end-joining by cell-free extract

Cited by 119 publications

References 46 publications

The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining

The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining

Eukaryotic DNA Ligases: Structural and Functional Insights

Ku Heterodimer-Independent End Joining inTrypanosoma bruceiCell Extracts Relies upon Sequence Microhomology

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