Functional analysis of XPE and XPD proteins in regard to the … · 2010-09-29 · Roc1. DDB1....

Functional analysis of XPE and XPD proteins in regard to the phenotypes of xeroderma pigmentosum patients

Kiyoji TanakaGraduate 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 TakedachiMasafumi Saijo

Takedachi et al., Mol Cell Biol, 2010

DDB1DDB2

XPCHR23B

PCNA

DNA pol ε or δ

RPA

XPGXPF

ERCC1

TFIIH

XPA

Nucleotide excision repair (NER)

Global genome NER(GGR)

Transcription-coupled NER(TCR)

RNA pol II

CyclobutanePyrimidine dimer

[CPD]

CH

3 NH2

O

N

O

O

N

N NH

H

H

H

(6-4) Photoproduct[(6-4)PP]

H

NH2

CH3

O

O

N

ONN

NH

UV-damaged DNA binding protein (UV-DDB)

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.

Cul4ARoc1

DDB1DDB2

E2

Cul4ARoc1

Ub

substrate

Ub

Ub E1

E2

E1E2Ub

E1Ub

ATP

ADP+Pi

+

Cul4ARoc1

DDB1DDB2

XPCHR23B


Cul4ARoc1

DDB1

XPCHR23B

UbUb

Ub

Ub

Ub

Cul4ARoc1

DDB1DDB2

UbCul4ARoc1

DDB1DDB2

Ub

Ub

“Hand-off from UV-DDB to the XPC complex at the damaged site.”

“Destabilization of chromatin structure”

DDB2

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.

E2

Cul4ARoc1

Ub

substrate

Ub

Ub E1

E2

E1E2Ub

E1Ub

ATP

ADP+Pi

+

Ub


Cul4ARoc1

DDB1DDB2

XPCHR23B

Ub

Cul4ARoc1

DDB1DDB2

Ub

Ub

“Hand-off from UV-DDB to the XPC/HR23B at the damaged site.”


Cul4ARoc1

DDB1

XPCHR23B

UbUb

UbDDB2

Mutant DDB2 derived from XP-E patients

Functions of the DDB2 E3 complex in the damage-recognition step of GGR

Regulatory mechanisms for the E3 activity of the DDB2 complex

Mutant DDB2 derived from XP-E patients

WT

K24

4E

anti-DDB1

anti-DDB2

anti-Cul4A

anti-Roc1

R27

3H

∆31

3-42

7

Vect

or

pcDNA3-FLAG-DDB2 WT / HeLaK244E

Whole cell extract

anti-FLAG IP

R273H∆313-427

<Purification of the DDB2 proteins>

The K244E DDB2 protein forms an E3 complex

Cul4ARoc1

DDB1DDB2

DDB2Exon No.

1 2 3 4 5 6 7 8 910

100 300 427 a.a.

…Nuclear localization signal …WT-40 repeat motif

K244E (A905G)

<mutation> <patient>

XP82TO200

R273H (G933A) XP2ROXP3RO

∆313-427 Ops1

WT

The K244E DDB2 complex displays E3 activityDDB2

Exon No.1 2 3 4 5 6 7 8 910

100 300 427 a.a.

…Nuclear localization signal …WT-40 repeat motif

K244E (A905G)

<mutation> <patient>

XP82TO200

R273H (G933A) XP2ROXP3RO

∆313-427 Ops1

WT

Cul4ARoc1

DDB1

WT

K24

4ER

273H

∆31

3-42

7

Vect

orB

uffe

r

WT

K24

4ER

273H

∆31

3-42

7

Vect

orB

uffe

rUb-DDB2DDB2

kDa250150100

755037

Poly-ubiquitinchain

kDa250150100

755037

DDB2UbUbiquitin ligase reaction (14µl)

50 mM

E3

Tris-HCl (pH 7.6)MgCl2DTTCaCl2ATP

UbcH5α; E2

UbiquitinE1

10 mM1 mM

0.2 mM4 mM5 µg

0.1 µg0.4 µg

kDa

WT

K24

4E

DDB1

DDB2

Cul4A

Roc1

CSN1CSN2CSN3CSN4CSN5CSN6CSN7

CSN8

250150100

75

50

37

25

15

20

Purification of the K244E DDB2 complexDDB2

Exon No.1 2 3 4 5 6 7 8 910

100 300 427 a.a.

…Nuclear localization signal…WT-40 repeat motif

K244E (A905G)<mutation>

<patient>XP82TO

pCAGGS-FLAG-HA-DDB2 WT / HeLa

Whole cell extract

anti-FLAG IP

anti-HA IP

<Purification of the DDB2 complexes>

WTDDB1

Cul4ARoc1

CSN

K244EDDB1

Cul4ARoc1

CSN

200

Silver staining

K244E

histones

anti-DDB1

anti-Cul4A

anti-HA (DDB2)

anti-Roc1

anti-H3

anti-H4

UbiquitinGST-UbiquitinE2 (UbcH5a)E2 (UbcH5a d.n.)Histone octamersReaction time (min)

The K244E DDB2 complex mediates histone ubiqitylation in vitro

kDa

250150100

755037

1520

10

kDa

250150100

755037

25

15

20

1025

1520

10

150100150100

75

Buf

fer

Moc

k

WT

K24

4E

+ +-

- +-

-- -

++ -- +

+ +- -

+-

+ +

+ ++ ++ + + + + --- -- -- - --- + +

++ ++ ++ + + + +-60 00 60 60

Buf

fer

Moc

k

WT

K24

4E

+ +-

- +-

-- -

++ -- +

+ +- -

+-

+ +

+ ++ ++ + + + + --- -- -- - --- + +

++ ++ ++ + + + +-60 00 60 60

Cul4ADDB1

Roc1DDB2

Ub

UbUb

Ub

DNA (UV-)DNA (UV+) -

--

--

-++

++

++

WT

K24

4E

Moc

k

anti-DDB1

anti-Cul4A

anti-HA (DDB2)

anti-CSN7

anti-Roc1

Unbound fr.Bound fr.


Unbound fr.

Bound fr.

Unbound fr.

Bound fr.

Unbound fr.

Bound fr.

The K244E DDB2 complex does not bind to UV-damaged DNA

Amplification of the biotinylated DNA (165 bp)

UV-irradiation (10 kJ/m2)

Incubation with DDB2 (WT or K244E) complex

Unbound fr. Bound fr.

Immobilization on beads

Streptavidinebeads

K244EDDB1

Cul4ARoc1

CSN

CSN

DNA (UV-)DNA (UV+) -

--

--

-++

++

++

WT

K24

4E

Moc

k

anti-DDB1

anti-Cul4A

anti-HA (DDB2)

anti-CSN7

anti-Roc1



Unbound fr.

Bound fr.

Unbound fr.

Bound fr.

Unbound fr.

Bound fr.

The K244E DDB2 complex does not bind to UV-damaged DNA

Amplification of the Biotinylated DNA (165 bp)

UV-irradiation (10 kJ/m2)

Incubation with DDB2 (WT or K244E) complex

Unbound fr. Bound fr.


Streptavidinebeads

Cul4ARoc1WTDDB1

Cul4ARoc1

The wild-type, but not K244E DDB2 complex is associated with core histones in a UV-dependent manner


UV-irradiation (20 J/m2, 0 min)

anti-FLAG IP

anti-HA IP

Solubilized chromatin fr.

- - -+ + +

WT

K24

4E

Moc

k

UVanti-DDB1anti-Cul4Aanti-HA (DDB2)anti-Roc1

anti-H2Aanti-H2Banti-H3anti-H4

K244E

The wild-type, but not K244E DDB2 complex is associated with core histones in a UV-dependent manner


UV-irradiation (20 J/m2)

anti-FLAG IP

anti-HA IP

Solubilizedchromatin fr. (C)

-WT K244E

0

DDB1

Cul4A

DDB2

Roc1H2AH2B

H3

H4

15 30 60 120 - 0 15 30 60 120NCNCNCNCNCNC NCNCNCNCNCNC

minfr.kDa

150

1007550

15

0, 15, 30, 60, 120 minNuclear extract (N)

K244E

Silver staining

Wild-type, but not K244E DDB2 is distributed to UV-damaged sites in chromatin in vivo

WT

K244E

UV-

UV+

UV-

UV+

anti-HA(DDB2) anti-XPC merge TO-PRO3pCAGGS-FLAG-HA-

DDB2 WT / HeLa

UV-irradiation (100 J/m2)[5-µm isopore filter]

Immunostaining

K244E

0 min

UV (100 J/m2)

5-µm isopore filter

Schematic representation of the strategy to measure thein vitro binding of DDB2 complex to UV-damaged chromatin

Mock or UV irradiated DNA

Mono-nucleosome reconstitution


Incubation with DDB2 complex

Supernatant(Unbound fr.)

Beads(Bound fr.)

In vitro ubiquitylation reaction

Flow-through fr. Released fr. Retained fr.

Streptavidinebeads


UV -- -- --+ ++ ++ +

Moc

k

anti-DDB1

anti-Cul4A

anti-HA (DDB2)

anti-CSN7

anti-Roc1

- - -+ + +

Flow-through RetainedReleased

WT

K24

4EM

ock

WT

K24

4EM

ock

WT

K24

4E

kDa 150100

3725

1520

250150100

250150100

755037

fr.

The wild-type, but not K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in vitro






Beads(Bound fr.)



CSN

Cul4ADDB1

Roc1K244E

Ub

UbUb

UV -- -- --+ ++ ++ +

Moc

k

anti-DDB1

anti-Cul4A

anti-HA (DDB2)

anti-CSN7

anti-Roc1

- - -+ + +

Flow-through RetainedReleased

WT

K24

4EM

ock

WT

K24

4EM

ock

WT

K24

4E

kDa 150100

3725

1520

250150100

250150100

755037

fr.







Beads(Bound fr.)



Cul4ARoc1

DDB1WT

Ub

UbUb

CSN

Cul4ADDB1

Roc1WT

UV-DDB1WT

Ub

Roc1Ub

Cul4A

Ub

Ub

UbUb

UV -- -- --+ ++ ++ +

Moc

k

anti-H2B

anti-H3

- - -+ + +

RetainedReleased

WT

K24

4EM

ock

WT

K24

4EM

ock

WT

K24

4E

kDa

37

25

15

20

250150100

7550

fr.

25

15

20

Flow-through







Beads(Bound fr.)



CSN

Cul4ADDB1

Roc1K244E

Ub

UbUb

UV -- -- --+ ++ ++ +

Moc

k

anti-H2B

anti-H3

- - -+ + +

RetainedReleased

WT

K24

4EM

ock

WT

K24

4EM

ock

WT

K24

4E

kDa

37

25

15

20

250150100

7550

fr.

25

15

20

Flow-through







Beads(Bound fr.)



Cul4ARoc1

Ub

Ub

DDB1WT

Ub

DDB1WT

Ub

CSNRoc1

Ub

Cul4A

Ub

Ub

ConclusionsBoth 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 nucleosomesbearing 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

Ub


Cul4ARoc1

DDB1DDB2

Ub

XPCHR23B

Ub

Cul4ARoc1

DDB1DDB2

Ub



Cul4ARoc1

DDB1

XPCHR23B

UbUb

UbDDB2

Ub


Cul4ARoc1

DDB1DDB2

Ub

XPCHR23B

Ub

Cul4ARoc1

DDB1DDB2

Ub



Cul4ARoc1

DDB1

XPCHR23B

UbUb

UbDDB2

Another possibility…

“The DDB2 complex-mediated ubiquitylation around damaged sites in chromatin may serve as a signal for the recruitment of other NER factors.”

The ectopically expressed wild-type DDB2 is colocalized with endogenous XPA

WT

K244E

UV-

UV+

UV-

UV+

anti-HA(DDB2) anti-XPA merge TO-PRO3pCAGGS-FLAG-HA-

DDB2 WT / HeLa


Immunostaining

K244E

0 min

UV (100 J/m2)

5-µm isopore filter

Schematic representation of the strategy


Beads

Retained fr.




Supernatant


Flow-through fr. Released fr.

III III Cul4ADDB1

Roc1DDB2

Ub

Ub

UbUb

Cul4ADDB1

Roc1DDB2

II

I

III


Beads

Retained fr.

Mono-nucleosome (UV- / UV+)

Mono-nucleosome (UV+)+DDB2 complex

Mono-nucleosome (UV+)+DDB2 complex+ubiquitylation

Incubation with

Flow-through fr. Bound fr.

XPC-HR23BorXPA

III

II

I

Cul4ARoc1

Ub

Ub37

25

1520

250150100

7550

25

1520

anti-H2B

anti-H3

anti-XPC

Inpu

tUV-

-+

+ -+-

++

+ +- +

+- +DDB2

Ub - -- +- -- +

fr.Flow-through Bound

anti-XPA

Inpu

tUV-

-+

+ -+-

++

+ +- +

+- +DDB2

Ub - -- +- -- +

Flow-through Boundfr.

The DDB2 complex-mediated ubiquitylation around damaged sites facilitates the recruitment of XPA

kDa

DDB1DDB2

Ub

Cul4ADDB1

Roc1DDB2

Ub

Conclusions

Implication for the DDB2 complex-mediated ubiquitylation in GGR

The DDB2 complex-mediated ubiquitylation around the damaged sites in chromatin functions in the signaling pathway during the recognition step in GGR.

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 nucleosomesbearing the UV-irradiated DNA, and consequently was not able to mediate the ubiquitylation of the nucleosomes assembled on UV-damaged DNA.

The DDB2 complex-mediated ubiquitylation around damaged sites in nucleosomesenhanced the recruitment of XPA to lesions.

Regulatory mechanisms for the E3 activity of the DDB2 complex

- - -+ + +

WT


anti-H2Aanti-H2B

anti-H3anti-H4

anti-XPC

The wild-type, but not K244E DDB2 complex interacts with the modified forms of XPC in a UV-dependent manner



anti-FLAG IP

anti-HA IP


K244E K24

4E

Moc

k

The wild-type, but not K244E DDB2 complex interacts with Ku in a UV-dependent manner



anti-FLAG IP

anti-HA IP


K244E

anti-Ku70anti-Ku86

- - -+ + +

WT


anti-H2Aanti-H2B

anti-H3anti-H4

anti-XPC

K24

4E

Moc

k

DD

B2

IPXP

G IP

anti-XPGanti-DDB2Nanti-Ku86anti-Ku70anti-MAT1anti-H3

Schematic representation of the strategy to measurethe effect of XPC/HR23B or Ku on the DDB2 E3 activity

UV irradiated DNA




Incubation with XPC-HR23B

In vitro ubiquitylation reaction (Ku -/+)

Released fr. Retained fr.

Cul4ADDB1

Roc1WT

XPCHR23B

Cul4ADDB1

Roc1WT

Nucleosome-free

WTDDB1

Cul4ARoc1

CSN

WTDDB1

Cul4ARoc1

CSN

XPCHR23B

Nucleosome-free

CSN

Cul4ADDB1

Roc1DDB2

XPCHR23B

Cul4ADDB1

Roc1DDB2

UV irradiated DNA







30 min reactionanti-HA (DDB2)

250150100

75

50

XPC stimulates the E3 activity of the DDB2 complex especially at damaged sites

anti-HA(DDB2)60 min reaction

250150100

75

50

kDa

++ -- --- -+ +- -+ - -- + -

RetainedReleased

-- ++ +++ +- -+ +- + ++ - +-- ++ --- -- -+ +- + -- - +-- -- ++- -+ ++ +- - +- + +

Ku70/86E2

d.n. E2

Nucleosome-freefr.XPC-HR23B

CSN

Cul4ADDB1

Roc1DDB2 Ub

Cul4ADDB1

Roc1DDB2

XPCHR23B

Ub

Ub

Ub

UV irradiated DNA







anti-Ku70100

75100kDa

++ -- --- -+ +- -+ - -- + -

RetainedReleased

-- ++ +++ +- -+ +- + ++ - +-- ++ --- -- -+ +- + -- - +-- -- ++- -+ ++ +- - +- + +

E2d.n. E2

Nucleosome-free

75anti-Ku86

fr.XPC-HR23B

Ku70/86

Cul4ADDB1

Roc1DDB2

XPCHR23B

Ku86Ku70

Cul4ADDB1

Roc1DDB2Ku86

Ku70

Ku inhibits the E3 activity of the DDB2 complex

Cul4ADDB1

Roc1DDB2

UV irradiated DNA







anti-HA(DDB2)

250150100

75

50

kDa

++ -- --- -+ +- -+ - -- + -

RetainedReleased

-- ++ +++ +- -+ +- + ++ - +-- ++ --- -- -+ +- + -- - +-- -- ++- -+ ++ +- - +- + +

E2d.n. E2

Nucleosome-free

Ku inhibits the E3 activity of the DDB2 complex

60 min reaction

fr.XPC-HR23B

Ku70/86

Nucleosome-free

CSN

Cul4ADDB1

Roc1DDB2Ub

Cul4ADDB1

Roc1DDB2

XPCHR23B

Ub

Ku86Ku70

Ub

Ku86Ku70

Ku86Ku70

anti-HA (DDB2)

kDa+ - -

RetainedReleasedfr.

++ - + -- + -- + - - +- - -+ + + - +Ku70/86

E2d.n. E2

15

20

250150100

75

50

100150

10anti-Roc1

10075

10075

10075

anti-DDB1

anti-Cul4A

anti-Ku70

anti-Ku86

Ku physically interacts with the DDB2 complex and acts as a negative regulator for the E3 activity

The DDB2 complex

Immobilization on anti-FLAG beads

In vitro ubiquitylation reaction (Ku- / +)


(FLAG-HA-DDB2 WT)

Cul4ADDB1

Roc1DDB2anti-FLAG

beads

anti-XPC

anti-HA(DDB2)

merge

TO-PRO3

si control si Ku86

0 min 15 minUV- UV+

0 min 15 minUV- UV+

sico

ntro

lsi

Ku8

6

anti-β-tubulinanti-Ku86

Ku affects the localization of DDB2to the damaged sites in vivo

pCAGGS-FLAG-HA-DDB2 / HeLa


Immunostaining

Transfection of siRNAs

ConclusionsBoth DNA-binding and E3 activities of the DDB2 complex are required to promote GGR in chromatin

Various regulatory mechanisms for the E3 activity of the DDB2 complex

Implication for the DDB2 complex-mediated ubiquitylation in GG-NER

The DDB2 complex-mediated ubiquitylation around the damaged sites in chromatin functions in the signaling pathway during the recognition step in GGR.

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 nucleosomesbearing the UV-irradiated DNA, and consequently was not able to mediate the ubiquitylation of the nucleosomes assembled on UV-damaged DNA.

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.

The DDB2 complex-mediated ubiquitylation around damaged sites in nucleosomesenhanced the recruitment of XPA to lesions.

Ku particularly inhibits the extensive autoubiquitylation of DDB2, and consequently prevents the dissociation of the DDB2 complex from damaged sites.

CSN

Model for the mechanism of damage recognition in GGR

Cul4ADDB1

Roc1WT

CSN

Cul4ARoc1

DDB1K244E

Cul4ADDB1

Roc1WT

XPCHR23B

Ku86Ku70

Cul4ADDB1

Roc1WT

XPCHR23B

Ub

Cul4A

DDB1

Roc1

WTKu86

Ku70

UbUb

Cul4A

Ub

Roc1Ub

DDB1WT

Ub

Ub Ub

XPA

Topics:The DDB2 complex-mediated ubiquitylation around DNA damage is oppositely regulated by XPC and Ku, and contributes to the recruitment of XPA



Shinsuke ItoLi Jing TanDaisuke AndohTakashi NaritaMineaki Seki

Ito S et al., Mol Cell, 2010

XP-AXP-BXP-CXP-DXP-EXP-FXP-GCS-ACS-BTTD-A

XP

XP

XP/CS, TTD

XPXP, XP/CSCSCSTTD

XPXP, XP/CS, TTD

Groups DisordersNER activity

UV-sensitivity GGR TCR Gene

＋＋＋＋＋

＋

＋＋

＋±

＋＋

＋

＋＋＋＋

＋

ーーーーーーー

ー

ーー

ーーーーーーー

XPAXPB(ERCC3)XPCXPD(ERCC2)XPE(DDB2 XPFXPG(ERCC5)CSA(ERCC8)CSB(ERCC6)TTDA

Genetic Complementation groups in XP, CS and TTD

(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 excisionrepair 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, andfacilitate optimal transcription. (S Dubaele et al., Nat Genet 20: 184, 1998; P Schultz etal., Cell 102: 599, 2000; B Sandrock et al., JBC 276: 35328, 2001; F Coin et al, Mol Cell11: 1635, 2003)

- a fraction of XPD is associated with CAK but not with the core TFIIH. (JT Reardon etal., Proc Natl Acad Sci USA 93: 6482, 1996)

-Drosophila Xpd was shown to negatively regulate CAK activity, resulting indecreased 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 (JChen et al., Nature 424: 228, 2003)Lack of Drosophila Xpd also cause defects in the dynamics of mitotic spindle andchromosomal 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-tubulinin 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 chromosomesegregation in MMS19-, MIP18-, and XPD-knockdown HCT116 cells

Blue: DAPI (chromosome)Green: alpha-tubulinRed: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

% fr

eque

ncy

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 MIP18exhibited 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 featuresof XP-D, XP-D/CS and XP-D/TTD patients

Frequencies of abnormal nuclei at inter-phase, and aberrant mitoticspindle and chromosomal segregation were examined in the cellsderived 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-2XPD/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 chromosome segregationAbnormal mitotic spindle

Abnormal nuclei in the interphase cells derivedfrom 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 XPDmutations 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

Photo: 長谷寺、奈良Hasedera Temple, Nara

Graduate School of Frontier Biosciences, Osaka UniversitySuita, Osaka, Japan

Yasushi HiraokaYasuhiro Hirano

AcknowledgementsJean-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

Functional analysis of XPE and XPD proteins in regard to the … · 2010-09-29 · Roc1. DDB1....

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