Proteome analysis of human metaphase chromosomes

Susumu Uchiyama, Shouhei Kobayashi, Hideaki Takata, Takeshi Ishihara, Naoto Hori, Tsunehito Higashi, Kayoko Hayashihara, Takefumi Sone, Daisuke Higo, Takashi Nirasawa, Toshifumi Takao, Sachihiro Matsunaga, Kiichi Fukui. Proteome Analysis of Human Metaphase Chromosomes. Journal of Biological Chemistry, Volume 280, Issue 17, 2005, Pages 16994-17004. https://doi.org/10.1074/jbc.M412774200

The middle region of an HP1-binding protein, HP1-BP74, associates with linker DNA at the entry/exit site of nucleosomal DNA

Kayoko Hayashihara, Susumu Uchiyama, Shigeru Shimamoto, Shouhei Kobayashi, Miroslav Tomschik, Hidekazu Wakamatsu, Daisuke No, Hiroki Sugahara, Naoto Hori, Masanori Noda, Tadayasu Ohkubo, Jordanka Zlatanova, Sachihiro Matsunaga, Kiichi Fukui. The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA. Journal of Biological Chemistry, Volume 285, Issue 9, 2010, Pages 6498-6507. https://doi.org/10.1074/jbc.M109.092833

The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA–Dependent Tel1 Activation

<div><p>Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.</p></div

Proposed molecular mechanism for promoting imprecise end joining in the <i>xrs2</i> FHA mutants.

<p>A. DSB repair process in <i>xrs2</i> FHA mutants. Compromised Tel1 activity causes accumulation or persistent localization of Ku at processed DSB ends, which then often facilitates Ku-dependent imprecise end joining. B. Tel1 is recruited to DSB ends in a manner dependent on Xrs2 Tel1-binding domain. C. Then, robust activation of Tel1 is promoted in an Xrs2 FHA-dependent manner. The color change and halo effect of Tel1 indicates relative amounts of Tel1 activation. D. Phosphorylation of Sae2 or other unknown factors by Tel1 and/or Mec1 plays a role in DSB end resection as well as Ku removal to promote HR.</p

Distribution of DSB repair types produced by non-complementary DSB repair.

<p>A. The assay system and classification of the repair type depend on the junction analysis. Repaired products generated by survivors shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005942#pgen.1005942.g001" target="_blank">Fig 1C</a> and others were classified into five categories. Category A: repaired by annealing of microhomology with large asymmetric resection of up to 60 nucleotides in a Ku- and DNA ligase IV (Lig4)-independent manner. Category B: repaired by end joining mediated by annealing of microhomology with resection. Category C: repaired by end joining without microhomology-mediated annealing at resected ends. Category D: repaired by end joining without microhomology-mediated annealing and end resection. Category E: repaired by end joining with microhomology-mediated annealing without resection. Categories B–E are Ku and DNA ligase IV dependent. B. Distribution of each category of repair type in various strains. Left: Frequency of each category shown in A of the imprecise–end joining categories in wild type (SLY19), <i>xrs2-SH</i> (DIY016), <i>tel1-KN</i> (MSY4629), <i>xrs2-664</i> (MSY4835), <i>xrs2</i>Δ (DIY007) and <i>yku70</i>Δ (DIY033). Numerical values are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005942#pgen.1005942.s007" target="_blank">S3 Table</a>. Right: Summary of classification of each category. n, number of samples analyzed for each strain.</p

Imprecise end joining increased in cells with Xrs2 FHA domain mutations in a Tel1-dependent manner.

<p>A. System used to examine imprecise end joining. Two HO-cutting sites were introduced in opposite orientations within the <i>MAT</i> locus on chromosome III, that result in two kinds of DSB ends: non-complementary single-stranded over hangs (Ura⁻) and complementary overhangs (Ura⁺), which are repaired by imprecise end joining and precise end joining, respectively. B. Xrs2 protein domains for wild-type and mutant <i>Saccharomyces cerevisiae</i> (sc) proteins. Mre11 BD, Mre11-binding domain; Tel1 BD, Tel1-binding domain. C. Frequencies of Ura⁻ and Ura⁺ cells indicate imprecise end joining and precise end joining, respectively, in wild-type (SLY19), <i>xrs2-314M</i> (DIY002), <i>xrs2-SH</i> (DIY016) and <i>xrs2</i>Δ (DIY007) strains. D. End-joining frequencies in wild-type, <i>tel1</i>Δ (DIY129), <i>tel1</i>Δ <i>xrs2-314M</i> (DIY131) and <i>tel1</i>Δ <i>xrs2-SH</i> (DIY134) strains assessed as in C. E. End-joining frequencies in wild-type, <i>sae2</i>Δ (DIY059), <i>sae2</i>Δ <i>xrs2-314M</i> (DIY062) and <i>sae2</i>Δ <i>xrs2-SH</i> (DIY065) strains assessed as in C. F. End-joining frequencies in wild-type (SLY19), <i>yku70</i>Δ (DIY033), <i>yku70</i>Δ <i>xrs2-314M</i> (DIY051) and <i>yku70</i>Δ <i>xrs2-SH</i> (DIY048) strains assessed as in C. G. End-joining frequencies in wild-type, <i>xrs2-SH</i> (DIY134), <i>lif1-SST</i> (DIY027), <i>lif1-T113A</i> (DIY072), <i>lif1-SST xrs2-SH</i> (MSY5655), <i>lif1-T113A xrs2-SH</i> (MSY5652) and <i>lif1</i>Δ (KMY691) strains assessed as in C. The wild-type data in C, D and E are from a single experiment. Data are presented as the mean ± SD from three independent experiments. Significance was calculated with a Student’s <i>t</i>-test: **<i>p</i><0.001; *<i>p</i><0.05; n.s., not significant.</p

An FHA mutation suppresses Tel1 activity but not Mec1 activity during both mitosis and meiosis.

<p>A. Phosphorylation of Rad53 protein was analyzed at the indicated time points after phleomycin addition. Rad53 protein was detected by Western blotting by using anti-Rad53 in wild type (W303-1A), <i>xrs2-SH</i> (MSY2199), <i>rad50S</i> (MSY2203), <i>rad50S mec1</i>Δ (MSY2461) and <i>mec1</i>Δ <i>rad50S xrs2-SH</i> (MSY2372). Asterisks indicate phosphorylation signal of Rad53. Tubulin blots are shown as internal control for protein loading. B. Meiosis progression at the indicated time points after SPM transition in wild type (NKY1551), <i>rad50S</i> (MSY1758), <i>xrs2-SH</i> (MSY1867), <i>rad50S xrs2-SH</i> (MSY1844), <i>xrs2-664</i> (MSY2015), <i>rad50S xrs2-664</i> (MSY2106) and <i>rad50S tel1</i>Δ (MSY1952). The percentage of cells containing two or more nuclei per ascus (post-MI%) was plotted in the graphs. Images to the right show typical DAPI images after 6 hr of meiosis in each cell line, and the corresponding percentage of post-MI cells for each strain is shown. Scale bar indicates 2 μm. C. Meiosis progression in wild type (NKY1551), <i>xrs2-SH</i> (MSY1867), <i>dmc1</i>Δ (MSY2638) and <i>dmc1</i>Δ <i>xrs2-SH</i> (MSY4674). Typical DAPI micrographs after 6 hr of meiosis in each cell line, with addition of <i>mec1Δ dmc1</i>Δ as a control, are shown to the right. Scale bar indicates 2 μm. D. Phosphorylation of Hop1 at T318 (pT318) at the indicated time points after SPM transition was analyzed in wild type (NKY1551), <i>rad50S</i> (MSY1758), <i>xrs2-SH</i> (MSY1867), <i>rad50S xrs2-SH</i> (MSY1844), <i>xrs2-664</i> (MSY2015), <i>rad50S xrs2-664</i> (MSY2106) and <i>rad50S tel1</i>Δ (MSY1952). Hop1 protein was analyzed by western blot with anti-Hop1 (Hop1; green) or with anti-Hop1-pT318 antibody (pT318; magenta), and then each fluorescence signal was detected by laser scanner on the same membrane. Highly mobility shifted band was marked with asterisk. A merged image is shown on the top. Tubulin blots are shown as an internal control for protein loading. E. Localization of Hop1 (red) and Hop1-pT318 (green) on the meiotic nuclear spreads at 4 hr after SPM transition was analyzed in wild type (NKY1551), <i>rad50S</i> (MSY1758), <i>xrs2-SH</i> (MSY1867), <i>rad50S xrs2-SH</i> (MSY1844), <i>xrs2-664</i> (MSY2015), <i>rad50S xrs2-664</i> (MSY2106) and <i>rad50S tel1</i>Δ (MSY1952). Scale bar indicates 2 μm.</p

Recruitment of DDR proteins to non-complementary DSB ends.

<p>A. Construction of DSB sites at the MAT locus on chromosome III of strain SLY19 and position of the primer pair for qPCR for ChIP analysis and QAOS assay. B. The association of Xrs2 protein with DSBs in SLY19 (–Tag) or in its derivatives with <i>XRS2-13Myc</i> (wild type, DIY109; <i>xrs2-314M</i>, DIY116; <i>xrs2-SH</i>, DIY106). Error bars show the SD from three independent qPCR trials. C. Time course analysis of the association of yKu70 protein with DSBs in SLY19 (–Tag) or in its derivatives with <i>YKU70-3FLAG</i> (wild type, MSY4829; <i>tel1</i>Δ, DIY118; <i>xrs2-SH</i>, DIY120). Data are presented as the mean ± SD from four independent trials. Significance was calculated with a Student’s <i>t</i>-test: **<i>p</i> < 0.001; *<i>p</i> < 0.05; n.s., not significant. D. The QAOS method is shown (upper). ssDNA at DSB ends was detected by QAOS assay in wild type (SLY19), xrs2-SH (DIY016), <i>tel1</i>Δ (DIY129) and <i>sae2</i>Δ (DIY059) cells. Data are presented as the mean ± SD from three independent experiments. Significance was calculated with a Student’s <i>t</i>-test: **<i>p</i> < 0.001; *<i>p</i> < 0.05; n.s., not significant. E. The assembly of Tel1 protein on DSBs in SLY19 (–Tag) or in its derivatives with <i>3FLAG-TEL1</i> (wild type, MSY5505; <i>xrs2-314M</i>, MSY5539; <i>xrs2-SH</i>, MSY5486; <i>xrs2-664</i>, MSY5541). F. Association of Sae2 protein with DSBs in SLY19 (–Tag) or in its derivatives with <i>SAE2-3HA</i> (wild type, DIY142; <i>xrs2-314M</i>, DIY144; <i>xrs2-SH</i>, DIY146). In B and F or E, a ChIP assay was performed at the HO cutting site at 150 or 120 min, respectively, after HO induction (DSB +) or before induction (DSB–). Error bars in C, E and F show the SD from three or more independent experiments, each of which consisted of an average from three independent qPCR trials for each strain.</p

RBMX: A Regulator for Maintenance and Centromeric Protection of Sister Chromatid Cohesion

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids