Functional analysis of XPE and XPD proteins in regard to the phenotypes of xeroderma pigmentosum patients
Kiyoji Tanaka Graduate School of Frontier Biosciences Osaka University
Videoconference for the DNA Repair Interest Group NIH, September 21, 2010 Photo: 祇王寺、京都 Gi-ohji Temple, Kyoto Topics:
The DDB2 complex-mediated ubiquitylation around DNA damage is oppositely regulated by XPC and Ku, and contributes to the recruitment of XPA
MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation The DDB2 complex-mediated ubiquitylation around DNA damage is oppositely regulated by XPC and Ku, and contributes to the recruitment of XPA
Arato Takedachi Masafumi Saijo
Takedachi et al., Mol Cell Biol, 2010 Nucleotide excision repair (NER)
Global genome NER Transcription-coupled NER (GGR) (TCR)
DDB1 DDB2 RNA pol II
XPC HR23B
TFIIH XPF Cyclobutane XPG (6-4) Photoproduct ERCC1 XPA Pyrimidine dimer [(6-4)PP] [CPD] RPA O
3 NH2 H CH3 O N CH H NH2 H N N N O O N H O N N O DNA pol ε or δ H H N
PCNA UV-damaged DNA binding protein (UV-DDB) Ub + E1 ATP DDB1 ADP+Pi Cul4A E1 DDB2 Ub Cul4A Roc1 substrate E2 Roc1 E2 E1 Ub Ub E2 Ub
UV-damaged-DNA binding protein (UV-DDB) is a heterodimer composed of two subunits, DDB1 and DDB2, and plays a key role in DNA damage recognition at an early stage of global genome nucleotide excision repair (GGR) pathway.
Mutations in DDB2 are responsible for xeroderma pigmentosum group E (XP-E).
UV-DDB is integrated in a complex including Cul4A and Roc1, and displays ubiquitin ligase (E3) activity. UV-damaged DNA binding protein (UV-DDB) Ub + E1 ATP Ub DDB1 ADP+Pi Cul4A E1 DDB2 Ub Cul4A Roc1 substrate E2 XPC Roc1 E2 E1 Ub Ub HR23B Ub E2 Ub Ub
Ub DDB1UV-damagedUb DDB1-DNA binding protein (UV-DDB) is a heterodimer composed Cul4A Cul4A DDB2of two subunits,DDB2 DDB1 and DDB2,“Hand and-off plays from a UVkey-DDB role toin theDNA XPC damage Roc1 Roc1 recognition at an early stage ofcomplex global genome at the damaged nucleotide site.” excision repair (GGR) pathway.XPC HR23B Ub Mutations in DDB2 are responsible for xeroderma pigmentosum group E (XP-E). DDB1 DDB2 Cul4A UV-DDB is integratedRoc1 in a complex including Cul4A and Roc1, and displays ubiquitin ligase (E3) activity.“Destabilization of chromatin structure” Ub
Ub UV-damaged DNA binding protein (UV-DDB)
Ub DDB1 DDB2 Cul4A Roc1 XPC Ub HR23B Ub
Ub DDB1 Cul4A DDB2 “Hand-off from UV-DDB to the XPC/HR23B Roc1 Mutant DDB2 derived from XP-E patients at the damaged site.” XPC HR23B FunctionsUb of the DDB2 E3 complex in the damage-recognition step of GGR DDB1 DDB2 Cul4A RegulatoryRoc1 mechanisms for the E3 activity of the“Destabilization DDB2 complex of chromatin structure” Ub
Ub Mutant DDB2 derived from XP-E patients DDB2 Exon
Vector 8 427 9 a.a. WT 10 WT
R273H
∆313-427 anti anti anti anti Ops1 XP82TO XP3RO XP2RO
…Nuclear localization signal …WT-40 repeat motif
DDB1 DDB2 Cul4A
313 - 427 Ub 313 - 427 Ubiquitin ligase reaction (14µl) Buffer Vector WT K244E R273H ∆ Roc1 Buffer Vector WT K244E R273H ∆ 50 mM Tris-HCl (pH 7.6) kDa kDa 10 mM MgCl2 Poly- 1 mM DTT 250 250 150 ubiquitin 150 0.2 mM CaCl2 4 mM ATP 100 chain 100 5 µg Ubiquitin 75 75 0.1 µg E1 50 50 Ub-DDB2 0.4 µg UbcH5α; E2 E3 37 37 DDB2 Purification of the K244E DDB2 complex DDB2 Exon No. Silver staining 1 2 3 4 5 6 7 8 910
100 200 300 427 a.a. WT K244E
Amplification of the
biotinylated DNA (165 bp) Mock WT K244E DNA (UV-) + - + - + - DNA (UV+) - + - + - + UV-irradiation (10 kJ/m2) Unbound fr. Bound fr. anti-DDB1 Incubation with DDB2 Unbound fr. (WT or K244E) complex Bound fr. anti-Cul4A Unbound fr. Immobilization on beads anti-HA Bound fr. (DDB2)
Unbound fr. Bound fr. Unbound fr.
Bound fr. anti-CSN7
Unbound fr.
Bound fr. Streptavidine anti-Roc1 beads The K244E DDB2 complex does not bind to UV-damaged DNA
Amplification of the
Biotinylated DNA (165 bp) Mock WT K244E DNA (UV-) + - + - + - DNA (UV+) - + - + - + UV-irradiation (10 kJ/m2) Unbound fr. Bound fr. anti-DDB1 Incubation with DDB2 Unbound fr. (WT or K244E) complex Bound fr. anti-Cul4A Unbound fr. Immobilization on beads anti-HA Bound fr. (DDB2)
Unbound fr. Bound fr. Unbound fr.
Bound fr. anti-CSN7 Cul4A Roc1CSN DDB1 Cul4A Unbound fr. DDB1 WT Roc1 K244E Cul4A CSN Bound fr. Roc1 Streptavidine anti-Roc1 beads The wild-type, but not K244E DDB2 complex is associated with core histones in a UV-dependent manner
pCAGGS-FLAG-HA-DDB2 WT / HeLa Mock WT K244E K244E UV - + - + - + UV-irradiation (20 J/m2, 0 min) anti-DDB1 anti-Cul4A Solubilized chromatin fr. anti-HA (DDB2) anti-Roc1 anti-FLAG IP anti-H2A anti-H2B anti-HA IP anti-H3 anti-H4 The wild-type, but not K244E DDB2 complex is associated with core histones in a UV-dependent manner
pCAGGS-FLAG-HA-DDB2 WT / HeLa K244E
UV-irradiation (20 J/m2) 0, 15, 30, 60, 120 min Silver staining Nuclear extract (N) WT K244E Solubilized min - 0 15 30 60 120 - 0 15 30 60 120 kDa fr.NCNCNCNCNCNC NCNCNCNCNCNC chromatin fr. (C) 150 DDB1 100 Cul4A anti-FLAG IP 75 50 DDB2 anti-HA IP 15 H3 H2A H2BRoc1 H4 Wild-type, but not K244E DDB2 is distributed to UV-damaged sites in chromatin in vivo anti-HA pCAGGS-FLAG-HA- (DDB2) anti-XPC merge TO-PRO3 DDB2 WT / HeLa K244E UV- UV-irradiation (100 J/m2) [5-µm isopore filter] WT 0 min UV+ Immunostaining
UV (100 J/m2) UV-
K244E
µ 5- m isopore filter UV+ Schematic representation of the strategy to measure the in vitro binding of DDB2 complex to UV-damaged chromatin
Mock or UV irradiated DNA Streptavidine beads
Mono-nucleosome reconstitution
Immobilization on beads
Incubation with DDB2 complex In vitro ubiquitylation reaction
Supernatant Beads (Unbound fr.) (Bound fr.)
In vitro ubiquitylation reaction
Flow-through fr. Released fr. Retained fr. The wild-type, but not K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in vitro Mock or UV irradiated DNA Flow- fr. through Released Retained Mono-nucleosome reconstitution
Immobilization on beads Mock WT K244E Mock WT K244E Mock WT K244E UV - + - + - + - + - + - + - + - + - + Incubation with DDB2 complex kDa 150 anti-DDB1 100 Supernatant Beads (Unbound fr.) (Bound fr.) 250 anti-Cul4A 150 In vitro ubiquitylation reaction 100
Flow-through fr. Released fr. Retained fr. 250 150 anti-HA 100 75 (DDB2) 50 CSN 37 37 DDB1 Ub anti-CSN7 Ub K244ECul4A 25 Roc1 20 Ub 15 anti-Roc1 The wild-type, but not K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in vitro Mock or UV irradiated DNA Flow- fr. through Released Retained Mono-nucleosome reconstitution
Immobilization on beads Mock WT K244E Mock WT K244E Mock WT K244E UV - + - + - + - + - + - + - + - + - + Incubation with DDB2 complex kDa 150 anti-DDB1 100 Supernatant Beads (Unbound fr.) (Bound fr.) 250 anti-Cul4A 150 In vitro ubiquitylation reaction 100
Flow-through fr. Released fr. Retained fr. Ub Ub 250 150 anti-HA DDB1 Cul4A 100 UV- 75 (DDB2) WT Roc1 Ub 50 CSN 37 Ub 37 DDB1 Ub Ub DDB1 anti-CSN7 Ub WT Cul4A WT Cul4A 25 Roc1 Roc1 20 Ub Ub 15 anti-Roc1 The wild-type, but not K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in vitro Mock or UV irradiated DNA Flow- fr. through Released Retained Mono-nucleosome reconstitution
Immobilization on beads Mock WT K244E Mock WT K244E Mock WT K244E UV - + - + - + - + - + - + - + - + - + Incubation with DDB2 complex kDa 25 Supernatant Beads 20 (Unbound fr.) (Bound fr.) 15 anti-H2B In vitro ubiquitylation reaction 250 150 Flow-through fr. Released fr. Retained fr. 100 75 50 37
CSN 25 DDB1 Ub 20 Ub anti-H3 K244ECul4A 15 Roc1 Ub The wild-type, but not K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in vitro Mock or UV irradiated DNA Flow- fr. through Released Retained Mono-nucleosome reconstitution
Immobilization on beads Mock WT K244E Mock WT K244E Mock WT K244E UV - + - + - + - + - + - + - + - + - + Incubation with DDB2 complex kDa 25 Supernatant Beads 20 (Unbound fr.) (Bound fr.) 15 anti-H2B In vitro ubiquitylation reaction 250 150 Flow-through fr. Released fr. Retained fr. 100 Ub Ub 75 DDB1 Cul4A 50 WT Roc1 37 Ub CSN 25 Ub Ub DDB1 20 anti-H3 WT Cul4A 15 Roc1 Ub Ub Conclusions Both DNA-binding and E3 activities of the DDB2 complex are required to promote GGR in chromatin K244E DDB2 was able to form the cullin-based E3 complex. However, this complex did not bind to nucleosomes in the UV-irradiated cells. The K244E DDB2 complex did not bind to the UV-damaged DNA or the nucleosomes bearing the UV-irradiated DNA, and consequently was not able to mediate the ubiquitylation of the nucleosomes assembled on UV-damaged DNA. Functions of the DDB2 E3 complex in the DNA damage-recognition step of GGR UV-damaged DNA binding protein (UV-DDB)
Ub DDB1 DDB2 Cul4A Roc1 XPC Ub HR23B Ub
Ub DDB1 Cul4A DDB2 “Hand-off from UV-DDB to the XPC Roc1 complex at the damaged site.” XPC HR23B Ub
DDB1 DDB2 Cul4A Roc1 “Destabilization of chromatin structure” Ub
Ub UV-damaged DNA binding protein (UV-DDB)
Ub DDB1 DDB2 Cul4A Roc1 XPC Ub HR23B Ub
Ub DDB1 Cul4A DDB2 “Hand-off from UV-DDB to the XPC AnotherRoc1 possibility… complex at the damaged site.” XPC “The DDB2 complexHR23B-mediated ubiquitylation around damaged sites in chromatin may Ubserve as a signal for the recruitment of other NER factors.”
DDB1 DDB2 Cul4A Roc1 “Destabilization of chromatin structure” Ub
Ub The ectopically expressed wild-type DDB2 is colocalized with endogenous XPA anti-HA pCAGGS-FLAG-HA- (DDB2) anti-XPA merge TO-PRO3 DDB2 WT / HeLa K244E UV- UV-irradiation (100 J/m2) [5-µm isopore filter] WT 0 min UV+ Immunostaining
UV (100 J/m2) UV-
K244E
µ 5- m isopore filter UV+ Schematic representation of the strategy Mock or UV irradiated DNA I Mono-nucleosome (UV- / UV+)
Mono-nucleosome reconstitution II Mono-nucleosome (UV+) +DDB2 complex
Immobilization on beads I III Mono-nucleosome (UV+) +DDB2 complex +ubiquitylation Incubation with DDB2 complex Incubation with XPC-HR23B or Supernatant Beads II XPA
In vitro ubiquitylation reaction Flow-through fr. Bound fr.
Flow-through fr. Released fr. Retained fr. III Ub Ub DDB1 I II III DDB1 DDB2 Cul4A DDB2 Cul4A Roc1 Roc1 Ub Ub The DDB2 complex-mediated ubiquitylation around damaged sites facilitates the recruitment of XPA
Flow- Flow- fr. through Bound fr. through Bound UV - + + + - + + + UV - + + + - + + + DDB2 - - + + - - + + DDB2 - - + + - - + +
Ub - - - + - - - + Input Ub - - - + - - - + Input anti-XPC kDa anti-XPA 25 20 DDB1 15 anti-H2B DDB2 Cul4A Roc1 250 150 100 75 50 Ub Ub DDB1 37 DDB2 Cul4A Roc1 25 Ub 20 Ub 15 anti-H3 Conclusions Both DNA-binding and E3 activities of the DDB2 complex are required to promote GGR in chromatin K244E DDB2 was able to form the cullin-based E3 complex. However, this complex did not bind to nucleosomes in the UV-irradiated cells. The K244E DDB2 complex did not bind to the UV-damaged DNA or the nucleosomes bearing the UV-irradiated DNA, and consequently was not able to mediate the ubiquitylation of the nucleosomes assembled on UV-damaged DNA. Implication for the DDB2 complex-mediated ubiquitylation in GGR The DDB2 complex-mediated ubiquitylation around damaged sites in nucleosomes enhanced the recruitment of XPA to lesions. The DDB2 complex-mediated ubiquitylation around the damaged sites in chromatin functions in the signaling pathway during the recognition step in GGR. Regulatory mechanisms for the E3 activity of the DDB2 complex The wild-type, but not K244E DDB2 complex interacts with the modified forms of XPC in a UV-dependent manner
pCAGGS-FLAG-HA-DDB2 WT / HeLa Mock K244E WT K244E UV - + - + - + UV-irradiation (20 J/m2, 0 min) anti-DDB1 anti-Cul4A Solubilized chromatin fr. anti-HA (DDB2) anti-Roc1 anti-FLAG IP anti-H2A anti-H2B anti-HA IP anti-H3 anti-H4
anti-XPC The wild-type, but not K244E DDB2 complex interacts with Ku in a UV-dependent manner
pCAGGS-FLAG-HA-DDB2 WT / HeLa Mock K244E WT K244E UV - + - + - + UV-irradiation (20 J/m2, 0 min) anti-DDB1 anti-Cul4A Solubilized chromatin fr. anti-HA (DDB2) anti-Roc1 anti-FLAG IP anti-H2A anti-H2B XPG IP anti-HA IP IPDDB2 anti-H3 anti-XPG anti-DDB2N anti-H4 anti-Ku86 anti-XPC anti-Ku70 anti-MAT1 anti-Ku70 anti-H3 anti-Ku86 Schematic representation of the strategy to measure the effect of XPC/HR23B or Ku on the DDB2 E3 activity
UV irradiated DNA
Mono-nucleosome reconstitution
Immobilization on beads DDB1 WT Cul4A Roc1 Incubation with DDB2 complex DDB1 WT Cul4A Roc1 Incubation with XPC-HR23B XPC HR23B Nucleosome-free In vitro ubiquitylation reaction (Ku -/+) CSN CSN DDB1 DDB1 Released fr. Retained fr. WT Cul4A WT Cul4A Roc1 Roc1 XPC HR23B XPC stimulates the E3 activity of the DDB2 complex especially at damaged sites UV irradiated DNA Mono-nucleosome reconstitution fr. Released Retained Nucleosome-free Immobilization on beads XPC-HR23B - - - + + + - - - + + + - - - + + + Ku70/86 - - + - - + - - + - - + - - + - - + Incubation with DDB2 complex E2 - ++ - + + - + + - + + - + + - + + Incubation with XPC-HR23B d.n. E2 + - - + - - + - - + - - + - - + - - In vitro ubiquitylation reaction (Ku -/+) kDa Released fr. Retained fr. 250 150 100 Ub 75 Ub DDB1 DDB1 DDB2 Cul4A DDB2 Cul4A 50 anti-HA Roc1 Roc1 XPC 60 min reaction (DDB2) HR23B Nucleosome-free 250 CSN CSN 150 100 DDB1 DDB1 Ub Ub 75 DDB2 Cul4A DDB2 Cul4A Roc1 Roc1 50 XPC anti-HA (DDB2) HR23B 30 min reaction Ku inhibits the E3 activity of the DDB2 complex
UV irradiated DNA Mono-nucleosome reconstitution fr. Released Retained Nucleosome-free Immobilization on beads XPC-HR23B - - - + + + - - - + + + - - - + + + Ku70/86 - - + - - + - - + - - + - - + - - + Incubation with DDB2 complex E2 - ++ - + + - + + - + + - + + - + + d.n. E2 + - - + - - + - - + - - + - - + - - Incubation with XPC-HR23B kDa100 75 anti-Ku70 In vitro ubiquitylation reaction (Ku -/+) 100 anti-Ku86 75 Released fr. Retained fr.
DDB1 DDB1 DDB2 Cul4A DDB2 Cul4A Ku86 Roc1 Ku86 Roc1 Ku70 Ku70 XPC HR23B Ku inhibits the E3 activity of the DDB2 complex
UV irradiated DNA Mono-nucleosome reconstitution fr. Released Retained Nucleosome-free Immobilization on beads XPC-HR23B - - - + + + - - - + + + - - - + + + Ku70/86 - - + - - + - - + - - + - - + - - + Incubation with DDB2 complex E2 - ++ - + + - + + - + + - + + - + + Incubation with XPC-HR23B d.n. E2 + - - + - - + - - + - - + - - + - - In vitro ubiquitylation reaction (Ku -/+) kDa Released fr. Retained fr. 250 150 100 Ub 75 Ub DDB1 DDB1 DDB2 Cul4A DDB2 Cul4A 50 anti-HA Ku86 Roc1 Ku86 Roc1 Ku70 Ku70 XPC 60 min reaction (DDB2) HR23B Nucleosome-free
CSN DDB1 Ub DDB2 Cul4A Ku86 Roc1 Ku70 Ku physically interacts with the DDB2 complex and acts as a negative regulator for the E3 activity fr. Released Retained Ku70/86 - + - + - + - + E2 - - + + - - - + The DDB2 complex(FLAG-HA-DDB2 WT) d.n. E2 + + - - + + - - kDa 150 100 anti-DDB1 Immobilization on anti-FLAG beads 100 75 anti-Cul4A
In vitro ubiquitylation reaction (Ku- / +) 250 150 Released fr. Retained fr. 100 75
50 anti-HA (DDB2) 20 DDB1 Cul4A 15 anti-FLAG DDB2 anti-Roc1 Roc1 10 beads 100 75 anti-Ku70 100 anti-Ku86 75 Ku affects the localization of DDB2 to the damaged sites in vivo pCAGGS-FLAG-HA-DDB2 / HeLa
si control si Ku86 Transfection of siRNAs UV- UV+ UV- UV+ 0 min 15 min 0 min 15 min UV-irradiation (100 J/m2) [5-µm isopore filter] anti-HA (DDB2) Immunostaining anti-XPC
merge control Ku86 si si anti-Ku86 TO-PRO3 anti-β-tubulin Conclusions Both DNA-binding and E3 activities of the DDB2 complex are required to promote GGR in chromatin K244E DDB2 was able to form the cullin-based E3 complex. However, this complex did not bind to nucleosomes in the UV-irradiated cells. The K244E DDB2 complex did not bind to the UV-damaged DNA or the nucleosomes bearing the UV-irradiated DNA, and consequently was not able to mediate the ubiquitylation of the nucleosomes assembled on UV-damaged DNA. Implication for the DDB2 complex-mediated ubiquitylation in GG-NER The DDB2 complex-mediated ubiquitylation around damaged sites in nucleosomes enhanced the recruitment of XPA to lesions. The DDB2 complex-mediated ubiquitylation around the damaged sites in chromatin functions in the signaling pathway during the recognition step in GGR. Various regulatory mechanisms for the E3 activity of the DDB2 complex CSN is released from the DDB2 complex when the complex binds to lesions. Correspondingly, the E3 activity of the DDB2 complex was stimulated. XPC stimulated the E3 activity of the DDB2 complex especially at damaged sites by stabilizing it. Ku negatively regulates the E3 activity of the DDB2 complex by destabilizing it. Ku particularly inhibits the extensive autoubiquitylation of DDB2, and consequently prevents the dissociation of the DDB2 complex from damaged sites. Model for the mechanism of damage recognition in GGR
CSN CSN DDB1 DDB1 K244E Cul4AWT Cul4A Roc1 Roc1
DDB1 DDB1 WT WT Cul4A Ku86 Cul4A Roc1 Ku70 XPC Ub Roc1 Ub DDB1 HR23B WT Cul4A Ku86 Roc1 Ku70 XPC Ub Ub HR23B Ub Ub Ub Cul4A DDB1 XPA Roc1 WT Ub Topics:
The DDB2 complex-mediated ubiquitylation around DNA damage is oppositely regulated by XPC and Ku, and contributes to the recruitment of XPA
MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation
Shinsuke Ito Li Jing Tan Daisuke Andoh Takashi Narita Mineaki Seki
Ito S et al., Mol Cell, 2010 Genetic Complementation groups in XP, CS and TTD
NER activity Groups Disorders Gene UV-sensitivity GGR TCR XP-A XP ＋＋＋ ー ー XPA XP-B XP/CS, TTD ＋＋ ー ー XPB(ERCC XP-C XP ＋ ー ＋ XPC3) XP-D XP, XP/CS, ＋＋ ー ー XPD(ERCC XP-E XPTTD ± ー ー XPE(DDB2) XP-F XP ＋ ー ー XPF2 XP-G XP, XP/CS ＋＋ ー ー XPG(ERCC CS-A CS ＋ ＋ ー CSA(ERCC5) CS-B CS ＋ ＋ ー CSB(ERCC8) 6) TTD-A TTD ＋ ー ー TTDA(p8) Multi-functions of XPD protein
- is one of the components of TFIIH, which has roles in nucleotide excision repair, basal transcription, transactivation and possibly cell cycle control.
- has a 5’ – 3’ DNA helicase and ATPase activities essential for nucleotide excision repair but dispensable for transcription. (F Coin et al., Mol Cell 26: 245, 2007)
- directly interacts with p44 and MAT1, serves as molecular bridge of holo-TFIIH, and facilitate optimal transcription. (S Dubaele et al., Nat Genet 20: 184, 1998; P Schultz et al., Cell 102: 599, 2000; B Sandrock et al., JBC 276: 35328, 2001; F Coin et al, Mol Cell 11: 1635, 2003)
- a fraction of XPD is associated with CAK but not with the core TFIIH. (JT Reardon et al., Proc Natl Acad Sci USA 93: 6482, 1996)
-Drosophila Xpd was shown to negatively regulate CAK activity, resulting in decreased phosphorylation of the cdk1 T-loop and inhibition of mitotic progression, while Xpd expression was down-regulated at G2/M when cdk1 was most active (J Chen et al., Nature 424: 228, 2003) Lack of Drosophila Xpd also cause defects in the dynamics of mitotic spindle and chromosomal instability, subcellular re-localizing of Cdk7 (X Li et al., Plos Genetics 6: e1000876, 2010) MMS19 and XPD complexes
MMS19 complex (MMXD)
XPD complex (XPG-TFIIH) Knockdown of MMS19 and MIP18 in HeLa cells
* Non-specific band MIP18 can directly interact with MMS19 and XPD MMS19 interacts with XPD in the region between the MAT1- and p44-binding domains (aa 438–637) MMXD (MMS19-MIP18-XPD) complex Co-localization of MIP18, MMS19 and GFP-XPD with alpha-tubulin in the mitotic phase of HCT116 cells
Scale bar: 10 μm Co-localization of GFP-XPD and MMS19 with alpha-tubulin, but not XPB in the mitotic phase of HCT116 cells
scale bar: 10μm Enhanced frequency of abnormal mitotic spindle and chromosome segregation in MMS19-, MIP18-, and XPD-knockdown HCT116 cells
Blue: DAPI (chromosome) Green: alpha-tubulin Red:cy3-prelabeled siRNA Abnormal localization of Aurora B in the MMS19- and MIP18-knockdown HeLa cells Enhanced frequency of abnormal nuclei in MMS19-, MIP18-, and XPD-knockdown HCT116 cells % frequency % FAM96B is designated MIP18 (MMS19-interacting protein 18 kDa) Possible functions of the MMXD protein complex in chromosome segregation
Cohesion between sister chromatids Formation of centrosome and/or mitotic spindles. Spindle checkpoint
Yeast yhr122w cells mutated in the putative yeast counterpart of MIP18 exhibited a chromosome instability phenotype, which is mostly associated with a defect in the establishment of sister chromatid cohesion.
Subunits of cohesins and condensins harbor HEAT repeats, which are frequently found in proteins required for chromosome dynamics including chromosome segregation, chromatin remodeling, and DNA repair. MMS19 contains HEAT repeat. It is plausible that the HEAT repeat in MMS19 functions with cohesins and condensins. Relevance of MMXD-dysfunction to clinical features of XP-D, XP-D/CS and XP-D/TTD patients
Frequencies of abnormal nuclei at inter-phase, and aberrant mitotic spindle and chromosomal segregation were examined in the cells derived from patients with XPD mutations.
XPD-patients’ cells: XP6BE (XP-D) XPCS-2 (XP-D/CS) TTD1RO (XP-D/TTD)
Wild-type XPD cDNA-corrected isogenic cells: XPD/XP6BE XPD/XPCS-2 XPD/TTD1RO Abnormal mitotic spindle and chromosome segregation in the metaphase cells derived from XP-D and XP-D/CS patients
Green: gamma-tubulin Red: alpha-tubulin Blue: DAPI Abnormal mitotic spindle and chromosome segregation in the metaphase cells derived from XP-D, XP-D/CS and XP-D/TTD patients
Abnormal mitotic spindle Abnormal chromosome segregation Abnormal nuclei in the interphase cells derived from XP-D(XP6BE) and XP-D/CS(XPCS-2) patients
Green: gamma-tubulin Blue: DAPI Abnormal nuclei in the interphase cells derived from XP-D, XP-D/CS and XP-D/TTD patients Clinical implication of MMXD-dysfunction:
The results indicate that the function of MMXD in chromosome segregation was affected in XP6BE (XP-D) and XPCS-2 (XP-D/CS) patients’ cells, but not in TTD1RO (XP-D/TTD) patient cells.
It is worth noting that these three patients had defects in NER, while skin tumors were detected only in XP6BE and XPCS-2 patients, not in TTD1RO patient.
These findings suggest that the dysfunction of MMXD due to the XPD mutations lead to aneuploidy and/or cell death, contributing at least partly to the phenotypes in XP6BE and XPCS-2 patients such as a high incidence of skin tumors. Collaboration
Graduate School of Frontier Biosciences, Osaka University Suita, Osaka, Japan
Yasushi Hiraoka Yasuhiro Hirano
Photo: 長谷寺、奈良 Hasedera Temple, Nara Acknowledgements
Jean-Marc Egly (IGBMC, France)
Wim Vermeulen (Erasmus University, The Netherlands)
Takeshi Horio (Kansai Medical University, Japan)
Errol C. Friedberg (University of Texas, USA)
Cherry Blossom, Osaka