Catherine L. Bladen

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).

BACKGROUND: Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population. OBJECTIVE: To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients. METHODS: In this cross-sectional study we analysed clinical data from 5345 genetically confirmed DMD patients from 31 countries held within the TREAT-NMD global DMD database. For analysis patients were categorised by corticosteroid background and further stratified by age. RESULTS: Loss of ambulation in non-steroid treated patients was 10 years and in corticosteroid treated patients 13 years old (p = 0.0001). Corticosteroid treated patients were less likely to need scoliosis surgery (p < 0.001) or ventilatory support (p < 0.001) and there was a mild cardioprotective effect of corticosteroids in the patient population aged 20 years and older (p = 0.0035). Patients with a single deletion of exon 45 showed an increased survival in contrast to other single exon deletions. CONCLUSIONS: This study provides data on clinical outcomes of DMD across many healthcare settings and including a sizeable cohort of older patients. Our data confirm the benefits of corticosteroid treatment on ambulation, need for scoliosis surgery, ventilation and, to a lesser extent, cardiomyopathy. This study underlines the importance of data collection via patient registries and the critical role of multi-centre collaboration in the rare disease field.

The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF·p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF·p54(nrb) complex cooperate to align separate DNA molecules. The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF·p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF·p54(nrb) complex cooperate to align separate DNA molecules. Living organisms are exposed to ionizing radiation from many sources. Biological effects of ionizing radiation include cell death, mutation, and transformation. The principal radiation target is DNA, and the most potent DNA lesions are double-strand breaks (DSBs), 1The abbreviations used are: DSB, double-strand break; PSF, protein-associated splicing factor; RRM, RNA recognition motif; NHEJ, nonhomologous end joining; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; mAb, monoclonal antibody; hnRNP, heterogenous nuclear ribonucleoprotein. caused when an ionization track creates clustered damage that affects both strands (1Hall E.J. Radiobiology for the Radiologist. 5th Ed. Lippincott, Williams and Wilkins, Philadelphia2000: 17-31Google Scholar). DSBs disrupt the physical integrity of the chromosome. If not repaired prior to cell division, they are often fatal to the cell. Also, incorrect joining of DSBs creates translocations and other chromosomal aberrations, leading to genetic instability, oncogene activation, and cancer. A main pathway of DSB repair in mammals is nonhomologous end joining (NHEJ). At least five polypeptides participate in mammalian NHEJ (reviewed in Refs. 2Lieber M.R. Ma Y. Pannicke U. Schwarz K. DNA Repair. 2004; 3: 817-826Crossref PubMed Scopus (196) Google Scholar and 3Meek K. Gupta S. Ramsden D.A. Lees-Miller S.P. Immunol. Rev. 2004; 200: 132-141Crossref PubMed Scopus (180) Google Scholar). They include the two subunits of Ku, which bind to DNA ends; DNA ligase IV (L4) and XRCC4 (X4), which form a complex that catalyzes strand ligation; and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which regulates the reaction. It is likely that additional NHEJ factors remain to be discovered. The five known polypeptides are insufficient to reconstitute regulated, high efficiency DNA end joining in a cell-free reaction (4Huang J. Dynan W.S. Nucleic Acids Res. 2002; 30: 667-674Crossref PubMed Scopus (119) Google Scholar). The addition of a small amount of nuclear extract to such a reaction, however, greatly increases its efficiency, suggesting the presence of additional DSB repair factors in the extract. The factors may accelerate the reaction by aligning separate DNA ends; such activity is likely to be crucial in vivo to prevent diffusion and reassortment of ends when multiple DSBs occur simultaneously. Although Ku and DNA-PKcs can hold separate DNA molecules together in vitro, such complexes are only marginally stable (5Pang D. Yoo S. Dynan W.S. Jung M. Dritschilo A. Cancer Res. 1997; 57: 1412-1415PubMed Google Scholar, 6Ramsden D.A. Gellert M. EMBO J. 1998; 17: 609-614Crossref PubMed Scopus (249) Google Scholar, 7Cary R.B. Peterson S.R. Wang J. Bear D.G. Bradbury E.M. Chen D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4267-4272Crossref PubMed Scopus (227) Google Scholar, 8DeFazio L.G. Stansel R.M. Griffith J.D. Chu G. EMBO J. 2002; 21: 3192-3200Crossref PubMed Scopus (274) Google Scholar), suggesting the involvement of additional proteins. To identify the repair factors present in nuclear extracts, we established a functional assay in which biochemical fractions, derived from HeLa cell nuclear extracts, were tested for their ability to stimulate end joining in the presence of recombinant Ku and L4·X4. In previous work, we identified two different stimulatory fractions (4Huang J. Dynan W.S. Nucleic Acids Res. 2002; 30: 667-674Crossref PubMed Scopus (119) Google Scholar, 9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). One fraction contains a >500-kDa complex of human Mre11, Rad50, and NBS1 polypeptides. These proteins, and their Saccharomyces cerevisiae homologs, have previously been implicated in DSB repair (10de Jager M. van Noort J. van Gent D.C. Dekker C. Kanaar R. Wyman C. Mol. Cell. 2001; 8: 1129-1135Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 11Chen L. Trujillo K.M. Ramos W. Sung P. Tomkinson A.E. Mol. Cell. 2001; 8: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 12Manolis K.G. Nimmo E.R. Hartsuiker E. Carr A.M. Jeggo P.A. Allshire R.C. EMBO J. 2001; 20: 210-221Crossref PubMed Scopus (136) Google Scholar, 13Yamaguchi-Iwai Y. Sonoda E. Sasaki M.S. Morrison C. Haraguchi T. Hiraoka Y. Yamashita Y.M. Yagi T. Takata M. Price C. Kakazu N. Takeda S. EMBO J. 1999; 18: 6619-6629Crossref PubMed Scopus (244) Google Scholar). The other fraction contains a ∼200-kDa factor that does not cross-react with antibodies against any of a variety of candidate proteins previously implicated in DSB repair. Only the latter fraction is capable of cooperating with DNA-PKcs to establish a phosphorylation-regulated end joining reaction (9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The two factors appear to participate in alternative, parallel pathways of DNA ligase IV-dependent end joining. We report here the identification of the ∼200-kDa factor as a complex of polypyrimidine tract binding protein-associated splicing factor (PSF) and p54 nuclear RNA-binding protein (p54(nrb)). These related polypeptides each contain tandem RNA recognition motifs (RRMs), together with conserved, homologous flanking sequences (14Zhang W.W. Zhang L.X. Busch R.K. Farres J. Busch H. Biochem. J. 1993; 290: 267-272Crossref PubMed Scopus (48) Google Scholar, 15Dong B. Horowitz D.S. Kobayashi R. Krainer A.R. Nucleic Acids Res. 1993; 21: 4085-4092Crossref PubMed Scopus (143) Google Scholar). Previous studies have suggested multiple functions for the PSF·p54(nrb) complex, including DNA recombination and RNA synthesis, processing, and transport (Refs. 16Mathur M. Tucker P.W. Samuels H.H. Mol. Cell. Biol. 2001; 21: 2298-2311Crossref PubMed Scopus (153) Google Scholar, 17Zhang Z. Carmichael G.G. Cell. 2001; 106: 465-475Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 18Zolotukhin A.S. Michalowski D. Bear J. Smulevitch S.V. Traish A.M. Peng R. Patton J. Shatsky I.N. Felber B.K. Mol. Cell. Biol. 2003; 23: 6618-6630Crossref PubMed Scopus (111) Google Scholar, 19Akhmedov A.T. Lopez B.S. Nucleic Acids Res. 2000; 28: 3022-3030Crossref PubMed Scopus (60) Google Scholar; reviewed in Ref. 20Shav-Tal Y. Zipori D. FEBS Lett. 2002; 531: 109-114Crossref PubMed Scopus (289) Google Scholar). We show here that the PSF·p54(nrb) complex strongly stimulates DNA end joining in vitro, binds directly to the DNA substrates of the end joining reaction, and cooperates with Ku to establish a functional preligation complex. DNA End Joining Assays—DNA end joining assays contained recombinant L4·X4 complex (21Lee K.J. Huang J. Takeda Y. Dynan W.S. J. Biol. Chem. 2000; 275: 34787-34796Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and non-His-tagged Ku heterodimer (22Yoo S. Kimzey A. Dynan W.S. J. Biol. Chem. 1999; 274: 20034-20039Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Some reactions also contained DNA-PKcs, which was purified using an affinity column containing 5 mg of a C-terminal Ku80 peptide (KGS-GEEGGDVDDLLDMI) (23Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (271) Google Scholar). The column was equilibrated with buffer A (25 mm HEPES-KOH (pH 7.5), 10% glycerol, 1 mm dithiothreitol, 0.1 mm EDTA) containing 0.05 m KCl. Nuclear extracts from 12.5 liters of HeLa cell culture (4Huang J. Dynan W.S. Nucleic Acids Res. 2002; 30: 667-674Crossref PubMed Scopus (119) Google Scholar) were passed over the column, which was eluted with a 0.05 to 1 m KCl gradient in buffer A. DNA-PKcs-containing fractions were further purified using a 1-ml Mono S ion exchange column pre-equilibrated with buffer DB (0.1 m KOAc, 20 mm Tris-HCl (pH 7.9), 1 mm EDTA, 1 mm dithiothreitol, and 20% glycerol) and protease inhibitors (10 μg/ml phenylmethylsulfonyl fluoride and 1 μg/ml each of pepstatin A, soybean trypsin inhibitor, leupeptin, and aprotinin). The column was eluted with a 0.1 m to 0.5 m KOAc gradient in buffer DB. End joining reactions were performed in a volume of 20 μl and contained 50 mm triethanolamine-HCl, 10 mm Tris-HCl (pH 7.9), 65 mm KOAc, 0.25 mm EDTA, 0.5 mm dithiothreitol, 10% glycerol, 1.0 mm Mg(OAc)2, 100 ng/μl bovine serum albumin, 1 mm ATP, 0.5 ng/μl substrate DNA (BamHI-linearized pUC 19 plasmid, 5′ end-labeled with polynucleotide kinase and [γ-32P]ATP), and proteins as indicated in the figure legends. The reactions were assembled without DNA and preincubated for 5 min at 37 °C, DNA was added, and incubation was continued for 30 min at 37 °C. The products were analyzed as described (4Huang J. Dynan W.S. Nucleic Acids Res. 2002; 30: 667-674Crossref PubMed Scopus (119) Google Scholar). Purification of DNA End Joining Stimulatory Factors—The factors were purified using nuclear extract from 50 liters of HeLa cell culture. Heparin-agarose, Q-Sepharose (0.3 m KOAc eluate), and Superdex 200 chromatography were performed as described (9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Active fractions from the Superdex 200 column were pooled and loaded onto a 10-ml single-strand DNA-agarose column (GE Healthcare, Piscataway, NJ), and eluted with a linear gradient of 0.1 to 1 m KOAc in Buffer DB. The active fractions were pooled, loaded onto a 1-ml Mono S column, and the column was eluted with a linear gradient of 0.1 to 1 m KOAc in Buffer DB. The active fractions were pooled and dialyzed against 0.1 m KOAc in Buffer DB. Two-dimensional Difference Gel Electrophoresis and Mass Spectrometry—Proteins were subjected to controlled labeling at lysine residues with Cy3 or Cy5 dyes. A Cy3 and a Cy5 labeling reaction was mixed and subjected to co-electrophoresis. Separate images of Cy3 and Cy5 fluorescence were collected and compared to identify polypeptides that were differentially abundant in each fraction. Spots of interest were excised and subjected to in-gel tryptic digestion. The peptides were analyzed by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry. Immunoprecipitation—Coupled in vitro transcription-translation reactions were performed using the TnT in vitro transcription/translation kit (Promega, Madison, WI). Metabolic labeling was performed as described previously (24Takeda Y. Caudell P. Grady G. Wang G. Suwa A. Sharp G.C. Dynan W.S. Hardin J.A. J. Immunol. 1999; 163: 6269-6274PubMed Google Scholar). Immunoprecipitation was performed as described (24Takeda Y. Caudell P. Grady G. Wang G. Suwa A. Sharp G.C. Dynan W.S. Hardin J.A. J. Immunol. 1999; 163: 6269-6274PubMed Google Scholar) in IPP buffer (10 mm Tris (pH 7.4), 0.5 m NaCl, and 0.1% Nonidet P-40). For immunodepletion, protein A-Sepharose beads (GE Healthcare) were loaded with antibody (specified in the figure legends) overnight at 4 °C. The beads were washed with IPP buffer and then with Buffer DB. Q-Sepharose 0.3 m KOAc eluate was added (30 and incubation was continued for was used for end joining reactions (10 contained buffer DNA substrate as in end joining and PSF·p54(nrb) and Ku as indicated in the figure legends. complexes were by and by Purification and of a End Joining have described previously a biochemical assay for the identification of NHEJ factors (9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). active fractions were of which contains a ∼200-kDa factor that is not with any of a of candidate proteins (9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). of factor was performed as a on single-strand DNA-agarose and Mono S two polypeptides with to and 100 that with stimulatory activity We the of factor activity on the other NHEJ proteins in the reaction. with previous activity was with Ku and L4·X4 the used (9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the presence of the DNA end joining activity was on L4·X4 and the that the factor DNA ligase End joining was also on Ku and of that activity in the presence of Ku and the stimulatory factor together was the of the activity in reactions with protein 5 compared with of and that Ku and the new factor have different functions in the end joining reaction. DNA-PKcs was not for end joining in the the addition of DNA-PKcs to a of activity and The addition of DNA-PKcs also the reactions to a small of DNA-PKcs and other S.P. G.C. Cancer Res. 1999; Google Scholar). In the of DNA-PKcs, in the presence of and DNA-PKcs, it activity by and The are with previous using purified factor (9Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). They are also with a model Y.V. Q. Lees-Miller S.P. Meek K. Ramsden D.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) where DNA-PKcs regulates the end joining reaction by binding to and the DNA ends and is then by by DNA-PKcs in the of that the efficiency of is the addition of the inhibitor, together with DNA-PKcs, to a further in end joining of the as a of and polypeptides in the active fractions were analyzed by A and in are in the active fraction These in mass to the polypeptides in the The comparison of two with different in the M. 1997; 18: PubMed Scopus Google Scholar). The here was an fraction from the Mono S column, fraction Spots A and were abundant in fraction in the fraction and A of of the the of the A as a protein of 100 The that it as a with to the as a protein of It as both of which were to contain the tryptic Spots to polypeptides A and were excised and with and the products were analyzed by mass spectrometry. peptide was for both of the tryptic with the protein identified A as polypyrimidine binding and as Although a mass of it previously been to at a to 100 in M. Tucker P.W. Samuels H.H. Mol. Cell. Biol. 2001; 21: 2298-2311Crossref PubMed Scopus (153) Google Scholar). The of the two polypeptides are with the on the motifs identified in and p54 are in The and C-terminal of the two polypeptides are related with and polypeptides contain which are also present in a variety of other RNA and single-strand proteins (reviewed in Ref. J.D. Mol. Biol. 23: PubMed Scopus Google Scholar). The are a human splicing motif by and other proteins B. Horowitz D.S. Kobayashi R. Krainer A.R. Nucleic Acids Res. 1993; 21: 4085-4092Crossref PubMed Scopus (143) Google Scholar). A of and not by other motif to the and to the human splicing with a previously to R. D.C. Patton 2002; 8: PubMed Scopus Google Scholar). of antibodies for in an we and by in vitro and of A and or by labeling in human and were in vitro, monoclonal antibody against each only that with and and were antibody against each the other and was by We also that two products were in the in vitro and A, and and and These from or the of in the and have not been cell containing PSF·p54(nrb) were used in an reaction, a in to and a in to The of the polypeptides was in separate by not These a previous report that and form a stable complex in vivo and in vitro R. D.C. Patton 2002; 8: PubMed Scopus Google Scholar). were performed using antibodies to that the PSF·p54(nrb) complex was the active in the end-joining factor The PSF·p54(nrb) complex was purified to separate it from the other stimulatory activity that is present in cell extracts (4Huang J. Dynan W.S. Nucleic Acids Res. 2002; 30: 667-674Crossref PubMed Scopus (119) Google Scholar). to the Q-Sepharose 0.3 m KOAc eluate was subjected to with or end joining activity to and with The addition of purified PSF·p54(nrb) activity to the extracts and on extracts and The that stimulatory activity is to the PSF·p54(nrb) complex or in a PSF·p54(nrb) DNA binding of PSF·p54(nrb) were in an assay using the DNA as in the end joining assays The addition of of protein in complexes Ku a different complex with DNA The addition of a small amount of Ku with PSF·p54(nrb) in of complexes that in from with protein The were Ku was and PSF·p54(nrb) or The that Ku and PSF·p54(nrb) are capable of binding DNA both and To complexes by Ku, and DNA were functional in the pathway we preincubated of purified Ku and PSF·p54(nrb) with of two which in ends We of PSF·p54(nrb) in to that substrate in to a added The are in are the of products by each of two different substrates of products were with each substrate and activity was in reactions where PSF·p54(nrb) or Ku were and show the substrate The is by 10 and where a DNA substrate that was preincubated with PSF·p54(nrb) complex and Ku, was in to that was preincubated with Ku with PSF·p54(nrb) and Ku in of a preligation complex. A reaction was performed where both substrates were with PSF·p54(nrb) and Ku, then and A mixed of products was A was when the two DNA substrates were mixed at the were also performed where substrate was preincubated with PSF·p54(nrb) and the other with Ku with PSF·p54(nrb) in substrate and activity was in 10 and the PSF·p54(nrb) and Ku cooperate only when present in on the they not cooperate in We present of that establish the complex as a candidate DNA end-joining and p54 are principal of an end-joining stimulatory which was purified on the of its activity with prior its active of a stimulatory fraction with monoclonal antibodies to and p54 end joining was by the addition of purified PSF·p54(nrb) complex. The PSF·p54(nrb) complex with the other proteins known to participate in NHEJ in was on Ku and L4·X4 and was by DNA-PKcs PSF·p54(nrb) also a preligation complex with Ku and DNA Previous identified multiple functions for the PSF·p54(nrb) complex in the cell It binds small nuclear RNA and may participate in splicing R. D.C. Patton 2002; 8: PubMed Scopus Google Scholar). It also heterogenous nuclear and in the Z. Carmichael G.G. Cell. 2001; 106: 465-475Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 18Zolotukhin A.S. Michalowski D. Bear J. Smulevitch S.V. Traish A.M. Peng R. Patton J. Shatsky I.N. Felber B.K. Mol. Cell. Biol. 2003; 23: 6618-6630Crossref PubMed Scopus (111) Google Scholar), and it nuclear activity M. Tucker P.W. Samuels H.H. Mol. Cell. Biol. 2001; 21: 2298-2311Crossref PubMed Scopus (153) Google Scholar). We propose that the PSF·p54(nrb) complex an previously in for in RNA and NHEJ is by Ku protein which with of in vivo and by both W. H. D.J. G.G. L. PubMed Scopus Google Scholar) and K. Huang J. Dynan W.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) are also of proteins with functions in and or repair L. S. Cell. 1993; Full Text PDF PubMed Scopus (289) Google Scholar, R. A. Huang L. K. A. D. PubMed Scopus Google Scholar, E. S. van R. W. A. D. EMBO J. 1997; PubMed Scopus Google Scholar, T. 1997; 275: PubMed Scopus Google Scholar). previously by and L4·X4 is active on model substrates in the of stimulatory factors (21Lee K.J. Huang J. Takeda Y. Dynan W.S. J. Biol. Chem. 2000; 275: 34787-34796Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, P. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In activity on double-strand DNA is used here where DNA substrate is the of both the PSF·p54(nrb) and the stimulatory factors is to the of DNA ends by in a preligation complex. NHEJ mammalian to of radiation the of that of breaks simultaneously. Although repair it does not for L. G. J. Biol. PubMed Scopus Google Scholar). of a preligation complex may the crucial A model for PSF·p54(nrb) DNA ends is by the of tandem in a complex with single-strand DNA J. Zhang Y. L. Krainer A.R. R.M. 1999; PubMed Scopus Google Scholar). In tandem domains of two are in a of single-strand DNA the protein complex to the protein the of DNA Ref. J. Zhang Y. L. Krainer A.R. R.M. 1999; PubMed Scopus Google Scholar). The four domains of the PSF·p54(nrb) complex bind DNA in a In domains bind to single-strand In the model in we have PSF·p54(nrb) as binding to an single-strand to the DNA with Ku to an We have also PSF·p54(nrb) as binding in to two simultaneously. Although the model is with we that the of the complex to be A cell or model or to be In small of in human was with in a which the of such assays to radiation Y. and W. S. The of cell may of the multiple functions of of which are for A of DSB repair proteins for is with Ku in human and with the complex in other Y. Sonoda E. Sasaki M.S. Morrison C. Haraguchi T. Hiraoka Y. Yamashita Y.M. Yagi T. Takata M. Price C. Kakazu N. Takeda S. EMBO J. 1999; 18: 6619-6629Crossref PubMed Scopus (244) Google Scholar, G. M.S. M. A.R. A. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, J. S. L. A. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). as with other proteins, it may be to the of of and on of radiation We Carmichael for of and p54 for the for HeLa and the and Mass at the of for protein

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.