Identification of GpA72 as T-Kininogen I by tandem mass spectrometry analysis.

<p>Protein band corresponding to GpA72 was excised and protolysed with trypsin. Extracted peptides were analyzed by liquid chromatography Tandem mass spectrometry using an ion trap mass spectrometer (LCQ Deca XP, Finnigan) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107565#s2" target="_blank">Methods</a> section. Top panel shows CID spectrum that was matched to ⁶⁶DGAETLYSFK⁷⁵ of T-kininogen 1. Observed b- and y-ions are indicated. Whole protein sequence and the peptides identified by LC-Tandem MS (bold) are shown in the bottom panel. Peptide sequences identified that aid in distinguishing the T-Kininogen I from T-Kininogen II are underlined.</p

Amino acid content of GpA72.

<p>The protein was hydrolyzed with 6N HCl under vacuum at 110°C for 24 h. Amino acid analysis was performed by pre-column derivatization with phenylisothiocynate. The phenylthiocarbomoylamino acids were analyzed using a Pico Tag column (3.9×150 mm)on a Waters HPLC system, equipped with a 1525 binary pump and Waters 2996-photodiode-array (PDA) detector set at 254 nm. Numbers on the peaks are retention times in minutes.</p

Serum levels of T-kininogen I in rats injected with different proinflammatory agents.

<p>Representative native PAGE gel image of sera from rats with hind joint injections of the following: Lane 1: Liquid paraffin oil, Lane 2: Adjuvant containing H37Rv, Lane 3: Turpentine oil; Lane 4: Zymosan; Lane 5: Carrageenan and Lane 6: Collagen.</p

Comparison of the amino acid compositions of GpA72 and α-Cysteine protease inhibitor.

<p>Comparison of the amino acid compositions of GpA72 and α-Cysteine protease inhibitor.</p

Dnl4 mutations under study.

<p>(<b>A</b>) Location of mutations made in this study relative to the functional domains of <i>S. cerevisiae</i> Dnl4 (yDnl4). DBD, DNA binding domain; AdD, adenylation domain; OBD, oligonucleotide binding domain; BRCT, BRCA1 C-terminal repeat; black oval, point mutation; red cross, stop codon. (<b>B</b>) Multiple sequence alignments surrounding conserved mutated yDnl4 positions. hLig4, human DNA ligase IV; yCdc9, <i>S. cerevisiae</i> DNA ligase I; hLig1, human DNA ligase I; Chlorella, chlorella virus DNA ligase. Magenta, identical among all proteins; red, identical to yDnl4; blue, conserved relative to yDnl4. (<b>C</b>) DNA ligase catalytic active site showing a structural alignment of hLig1 bound to a 5′-adenylated DNA nick (PDB 1X9N <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003599#pgen.1003599-Pascal1" target="_blank">[5]</a>, shaded more lightly) and adenylated Chlorella virus ligase bound to a nick (PDB 2Q2T <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003599#pgen.1003599-Nair1" target="_blank">[22]</a>, shaded more darkly). Shown are the AMP (yellow), the substrate DNA strand with labeled 3′ and 5′ nick termini, and the universally conserved residues under study, labeled as the homologous positions in yDnl4. Protein and DNA are shaded by element.</p

<i>Saccharomyces cerevisiae</i> DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

<div><p>DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation <i>in vitro</i> and NHEJ activity <i>in vivo</i>, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that <i>dnl4</i>-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as <i>dnl4</i> point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.</p></div

HPLC elution profile of the purified fraction of GpA72 Sera from four to six arthritic rats were pooled and subjected to protein purification as described under the methods section.

<p>The single peak eluting at 21.225 min represented GpA72.</p

Catalytically inactive Dnl4 protein does not impede DSB resection.

<p>DSBs were induced in yeast strains bearing the <i>ILV1</i>-cs allele similarly to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003599#pgen-1003599-g003" target="_blank">Figure 3</a> and monitored by Southern blotting. (<b>A</b>) Blot showing that DSB resection is not observed without the addition of glucose at 60 min. (<b>B</b>) Example blot showing formation of the HO-cut band and its subsequent disappearance by a combination of NHEJ and DSB resection. (<b>C</b>) and (<b>E</b>) The ratio of the HO-uncut band to the <i>APN1</i> control was normalized to the ratio at time 0 to allow monitoring of DSB formation and repair by NHEJ. (<b>D</b>) and (<b>F</b>) The ratio of the HO-cut band to the <i>APN1</i> control was normalized to the ratio at 60 min when DSB formation was maximal. Disappearance of the HO-cut band at subsequent times results from NHEJ (wild-type strain only) and/or DSB resection. (<b>C</b>) and (<b>D</b>) show results from asynchronous cells while (<b>E</b>) and (<b>F</b>) show results from cells arrested in G1 with α-factor. Results are the mean ± standard deviation of five independent experiments.</p

End joining profiles after extensive HO recleavage depend on Dnl4 status.

<p>(<b>A</b>) Fractions of different <i>ILV1</i>-cs joint categories after 24 hours of HO induction, showing preserved imprecise joining with K466A. “Precise sequence” is the same as the input allele prior to DSB formation, “major imprecise joints” had a frequency ≥0.1% in any tested strain, and “other joints” are the remainder. (<b>B</b>) Individual major imprecise joints that showed significantly different frequencies between wild-type and K466A. The left half shows joints that had increased frequency in K466A as compared to wild-type. The right half shows joints that had a decreased frequency. Red bars, joint fraction in K466A; green bars, joint fraction in wild-type. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003599#pgen.1003599.s005" target="_blank">Figure S5</a> for a description of the joint identifiers and File S1 for their sequences.</p

Extensive recruitment of catalytically inactive Dnl4 and associated c-NHEJ factors to a chromosomal DSB.

<p>Yeast strains bearing the indicated Dnl4 mutations and the <i>GAL1</i>-cs allele were grown in galactose medium for 60 min to induce HO expression. Cells were then transferred to glucose to allow repair by NHEJ. (<b>A</b>) The fraction of intact <i>GAL1</i>-cs HO cut sites showing the extent of DSB formation and repair over time, determined by flanking PCR. (<b>B</b>) The corresponding enrichment of 13Myc-tagged Dnl4 at the <i>GAL1</i>-cs DSB relative to the <i>ACT1</i> control locus as determined by ChIP from the same samples as (A). (<b>C</b>) Enrichment of 13Myc-tagged Lif1 and (<b>D</b>) 13Myc-tagged Yku80 at the <i>GAL1</i>-cs DSB. Results are the mean ± standard deviation of at least two independent experiments.</p