#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Rad3 Decorates Critical Chromosomal Domains with γH2A to Protect Genome Integrity during S-Phase in Fission Yeast


Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund–Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.


Vyšlo v časopise: Rad3 Decorates Critical Chromosomal Domains with γH2A to Protect Genome Integrity during S-Phase in Fission Yeast. PLoS Genet 6(7): e32767. doi:10.1371/journal.pgen.1001032
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1001032

Souhrn

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund–Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.


Zdroje

1. AguileraA

Gomez-GonzalezB

2008

Genome instability: a mechanistic view of its causes and consequences.

Nat Rev Genet

9

204

217

2. BoddyMN

RussellP

2001

DNA replication checkpoint.

Curr Biol

11

R953

956

3. CimprichKA

CortezD

2008

ATR: an essential regulator of genome integrity.

Nat Rev Mol Cell Biol

9

616

627

4. MirkinE

MirkinS

2007

Replication fork stalling at natural impediments.

Microbiol and Mol Biol Rev

71

13

35

5. PearsonC

Nichol EdamuraK

ClearyJ

2005

Repeat instability: mechanisms of dynamic mutations.

Nat Rev Genet

6

729

742

6. RogakouEP

PilchDR

OrrA

IvanovaV

BonnerWM

1998

DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.

J Biol Chem

273

5858

5868

7. DownsJA

LowndesNF

JacksonSP

2000

A role for Saccharomyces cerevisae histone H2A in DNA repair.

Nature

408

1001

1004

8. NakamuraTM

DuLL

RedonC

RussellP

2004

Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast.

Mol Cell Biol

24

6215

6230

9. RogakouEP

BoonC

RedonC

BonnerWM

1999

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

J Cell Biol

146

905

916

10. KimJ

KruhlakM

DotiwalaF

NussenzweigA

HaberJE

2007

Heterochromatin in refractory to yH2AX modification in yeast and mammals.

J Cell Biol

178

209

218

11. BassingCH

2002

Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX.

PNAS

99

8173

8178

12. CelesteA

PetersenS

RomanienkoPJ

Fernandez-CapetilloO

ChenHT

2002

Genomic instability in mice lacking histone H2AX.

Science

296

922

927

13. MartiT

HefnerE

FeeneyL

NataleV

CleaverJ

2006

H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks.

PNAS

103

9891

9896

14. WardI

ChenJ

2001

Histone H2AX is phosphorylated in an ATR-dependent manner in response to replication stress.

J Biol Chem

276

42462

42467

15. CobbJ

SchlekerT

RojasV

BjergbaekL

TerceroJ

2005

Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.

Genes Dev

19

3055

3069

16. Fernandez-CapetilloO

LeeA

NussenzweigM

NussenzweigA

2004

H2AX: the histone guardian of the genome.

DNA Repair (Amst)

3

959

967

17. BoglioloM

LyakhovichA

CallenE

CastellaM

CapelliE

2007

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability.

EMBO J

26

1340

1351

18. Papamichos-ChronakisM

PetersonCL

2008

The Ino80 chromatin-remodeling enzyme regulates replisome function and stability.

Nat Struct Mol Biol

15

338

345

19. StuckiM

ClappertonJA

MohammadD

YaffeMB

SmerdonSJ

2005

MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks.

Cell

123

1213

1226

20. DuL-L

NakamuraTM

RussellP

2006

Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand breaks.

Genes Dev

20

1583

1596

21. WilliamsJS

WilliamsRS

DoveyCL

GuentherG

TainerJA

2010

gammaH2A binds Brc1 to maintain genome integrity during S-phase.

EMBO J

22. VerkadeH

BuggS

LindsayH

CarrA

O'ConnellMJ

1999

Rad18 is required for DNA repair and checkpoint responses in fission yeast.

Mol Bio Cell

10

2905

2918

23. SheedyDM

DimitrovaD

RankinJK

BassKL

LeeKM

2005

Brc1-mediated DNA repair and damage tolerance.

Genetics

171

457

468

24. RouseJ

2004

Esc4p, a new target of Mec1p (ATR), promotes resumption of DNA synthesis after DNA damage.

EMBO J

23

1188

1197

25. ChoEA

PrindleMJ

DresslerGR

2003

BRCT domain-containing protein PTIP is essential for progression through mitosis.

Mol Cell Biol

23

1666

1673

26. MeisterP

TaddeiA

VernisL

PoidevinM

GasserSM

2005

Temporal separation of replication and recombination requires the intra-S checkpoint.

J Cell Biol

168

537

544

27. CoulonS

GaillardPH

ChahwanC

McDonaldWH

YatesJR3rd

2004

Slx1-Slx4 are subunits of a structure-specific endonuclease that maintains ribosomal DNA in fission yeast.

Mol Biol Cell

15

71

80

28. StewartE

ChapmanC

Al-KhodairyF

CarrAM

EnochT

1997

rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest.

EMBO J

16

2682

2692

29. MurrayJM

LindsayHD

MundayCA

CarrAM

1997

Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance.

Mol Cell Biol

17

6868

6875

30. WinT

MankouriH

HicksonID

WangS-W

2005

A role for the fission yeast Rqh1 helicase in chromosome segregation.

J Cell Sci

118

5777

5784

31. SzilardRK

JacquesPE

LarameeL

ChengB

GaliciaS

2010

Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX.

Nat Struct Mol Biol

32. DuLL

NakamuraTM

MoserBA

RussellP

2003

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes.

Mol Cell Biol

23

6150

6158

33. RedonC

PilchDR

RogakouEP

OrrAH

LowndesNF

2003

Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage.

EMBO Rep

4

678

684

34. ShroffR

Arbel-EdenA

PilchDR

IraG

BonnerWM

2004

Distribution and dynamics of chromatin modification at a defined DNA double strand break.

Curr Biol

14

1703

1711

35. DalgaardJZ

KlarAJ

2001

A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S.pombe.

Genes Dev

15

2060

2068

36. RoseaulinL

YamadaY

TsutsuiY

RussellP

IwasakiH

2008

Mus81 is essential for sister chromatid recombination at broken replication forks.

EMBO J

27

1378

1387

37. ArcangioliB

1998

A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast.

EMBO J

10

3025

3032

38. KlarA

MiglioL

1986

Initiation of meiotic recombination by double-strand breaks in S.pombe.

Cell

46

725

731

39. ForsburgSL

2003

Growth and manipulation of S. pombe.

Curr Protoc Mol Biol Chapter 13: Unit 13

16

40. RussellP

NurseP

1986

cdc25+ functions as an inducer in the mitotic control of fission yeast.

Cell

45

145

153

41. JohnsonWE

LiW

MeyerCA

GottardoR

CarrollJS

2006

Model-based analysis of tiling-arrays for ChIP-chip.

PNAS

103

12457

12462

42. NakamuraTM

MoserBA

RussellP

2002

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres.

Genetics

161

1437

1452

43. MatsuuraA

NaitoT

IshikawaF

1999

Genetic control of telomere integrity in S. pombe: rad3(+) and tel1(+) are parts of two regulatory networks independent of the downstream protein kinases chk1(+) and cds1(+).

Genetics

152

1501

1512

44. WoodV

2002

The genome sequence of Schizosaccharomyces pombe.

Nature

415

871

880

45. NomaK

CamHP

MaraiaRJ

GrewalSI

2006

A role for TFIIIC transcription factor complex in genome organization.

Cell

125

859

872

46. StyrkársdóttirU

EgelR

NielsenO

1993

The smt-0 mutation which abolishes mating-type switching in fission yeast is a deletion.

Curr Genet

23

184

186

47. EgelR

2004

DNA replication: stalling a fork for imprinting and switching.

Curr Biol

14

R915

917

48. NoguchiE

NoguchiC

McDonaldW

YatesJIII

RussellP

2004

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast.

Mol Cell Biol

24

8342

8355

49. AhnJ

OsmanF

WhitbyM

2005

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast.

EMBO J

24

2011

2023

50. Sánchez-GorostiagaA

Lopez-EstranoC

KrimerD

SchvartzmanJ

HernándezP

2004

Transcription termination factor reb1p causes two replication fork barriers at its cognate sites in fission yeast ribosomal DNA in vivo.

Mol Cell Biol

24

398

406

51. KringsG

BastiaD

2004

Swi1- and swi3-dependent and independent replication fork arrest at the ribosomal DNA of Schizosaccharomyces pombe.

Proc Natl Acad Sci U S A

101

14085

14090

52. TsangE

CarrAM

2008

Replication fork arrest, recombination and the maintenance of ribosomal DNA stability.

DNA Repair (Amst)

53. KobayashiT

HeckD

NomuraM

HoriuchiT

1998

Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I.

Genes Dev

12

3821

3830

54. Mejía-RamírezE

Sánchez-GorostiagaA

KrimerD

SchvartzmanJ

HernándezP

2005

The mating type switch-activating protein Sap1 is required for replication fork arrest at the rRNA genes of fission yeast.

Mol Cell Biol

25

8755

8761

55. NoguchiE

NoguchiC

DuLL

RussellP

2003

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1.

Mol Cell Biol

23

7861

7874

56. PryceDW

RamayahS

JaendlingA

McFarlaneRJ

2009

Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1.

Proc Natl Acad Sci U S A

106

4770

4775

57. DeshpandeAM

NewlonCS

1996

DNA replication forks pause sites dependent on transcription.

Science

272

1030

1033

58. ScottK

WhiteC

WillardH

2007

An RNA Polymerase III-dependent heterochromatin barrier at fission yeast centromeres.

PLoS ONE

2

e1099

doi:10.1371/journal.pone.0001099

59. GrewalSI

ElginSC

2007

Transcription and RNA interference in the formation of heterochromatin.

Nature

447

399

406

60. VolpeT

KidnerC

HallI

TengG

GrewalS

2002

Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.

Science

297

1833

1837

61. CamHP

SugiyamaT

ChenES

ChenX

FitzGeraldPC

2005

Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.

Nat Genet

37

809

819

62. PidouxA

ChoiE

AbbottJ

LiuX

KaganskyA

2009

Fission yeast Scm3: a CENP-A receptor required for integrity of subkinetochore chromatin.

Mol Cell

33

299

311

63. WilliamsJ

HayashiT

YanagidaM

RussellP

2009

Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin.

Mol Cell

33

287

298

64. HansenKR

BurnsG

MataJ

VolpeTA

MartienssenRA

2005

Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

Mol Cell Biol

25

590

601

65. WirenM

SilversteinRA

SinhaI

WalfridssonJ

LeeA

2005

Genome-wide analysis of nucleosome density histone acetylation and HDAC function in fission yeast.

EMBO J

24

2906

2918

66. MataJ

LyneR

BurnsG

BahlerJ

2002

The transcriptional program of meiosis and sporulation in fission yeast.

Nat Genet

32

143

147

67. BowenN

JordaI

EpsteinJ

WoodV

LevinH

2003

Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements from the complete genome sequence of Schizosaccharomyces pombe.

Genome Res

13

1984

1997

68. LinderT

GustafssonCM

2008

Molecular phylogenetics of ascomycotal adhesins - a novel family of putative cell-surface adhesive proteins in fission yeasts.

Fugal Genet Biol

45

485

497

69. de la SernaIL

ImbalazanoAN

2002

Unfolding heterochromatin for replication.

Nature Genetics

32

560

562

70. GomezE

EspinosaJ

ForsburgS

2005

Schizosaccharomyces pombe mst2+ encodes a MYST family histone acetyltransferase that negatively regulates telomere silencing.

Mol Cell Biol

25

8887

8903

71. BernardP

MaureJ

PartridgeJ

GenierS

JaverzatJ

2001

Requirement of heterochromatin for cohesion at centromeres.

Science

294

2539

2542

72. StromL

LindroosH

ShirahigeK

SjogrenC

2004

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair.

Mol Cell

16

1003

1015

73. UnalE

Arbel-EdenA

SattlerU

ShroffR

LichtenM

2004

DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain.

Mol Cell

16

991

1002

74. HayashiMT

TakahashiTS

NakagawaT

NakayamaJ

MasukataH

2009

The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus.

Nat Cell Biol

11

357

362

75. CooperJP

NimmoER

AllshireRC

CechTR

1997

Regulation of telomere length and function by a Myb-domain protein in fission yeast.

Nature

385

744

747

76. MillerK

RogO

CooperJP

2006

Semi-conservative DNA replication through telomeres requires Taz1.

Nature

440

824

828

77. KanohJ

SadaieM

UranoT

IshikawaF

2005

Telomere binding protein Taz1 establishes Swi6 heterochromatin independently of RNAi at telomeres.

Curr Biol

15

1808

1819

78. ChaRS

KlecknerN

2002

ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones.

Science

297

602

606

79. CasperA

NghiemP

ArltM

GloverT

2002

ATR regulates fragile site stability.

Cell

111

779

789

80. GrewalSI

JiaS

2007

Heterochromatin revisited.

Nat Rev Genet

8

35

46

81. ChenE

ZhangK

NicolasE

CamH

ZofallM

2008

Cell cycle control of centromeric repeat transcription and heterochromatin assembly.

Nature

451

734

737

82. KlocA

ZarateguiM

NoraE

MartienssenR

2008

RNA interference guides histone modification during the S phase of chromosomal replication.

Curr Biol

18

490

495

83. AguileraA

2002

The connection between transcription and genomic instability.

EMBO J

21

195

201

84. GreenfederSA

NewlonCS

1992

Replication forks pause at yeast centromeres.

Mol Cell Biol

12

4056

4066

85. SmithJ

CaddleM

BulboacaG

WohlgemuthJ

BaumM

1995

Replication of centromere II of Schizosaccharomyces pombe.

Mol Cell Biol

15

5165

5172

86. NakamuraK

OkamotoA

KatouY

YadaniC

ShitandaT

2008

Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe.

EMBO J

27

3036

3046

87. PebernardS

SchafferL

CampbellD

HeadS

BoddyM

2008

Localization of Smc5/6 to centromeres and telomeres requires heterochromatin and SUMO, respectively.

EMBO J

27

3011

3023

88. ChadwickBP

LaneTF

2005

BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation.

Chromosoma

114

432

439

89. Torres-RosellJ

MachinF

AragonL

2005

Smc5-Smc6 complex preserves nucleolar integrity in S. cerevisiae.

Cell Cycle

4

868

872

90. PebernardS

McDonaldWH

PavlovaY

YatesJR3rd

BoddyMN

2004

Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis.

Mol Biol Cell

15

4866

4876

91. Torres-RosellJ

MachinF

FarmerS

JarmuzA

EydmannT

2005

SMC5 and SMC6 genes are required for the segregation of repetitive chromosome regions.

Nat Cell Biol

7

412

419

92. ChinJK

BashkirovVI

HeyerWD

RomesbergFE

2006

Esc4/Rtt107 and the control of recombination during replication.

DNA Repair (Amst)

5

618

628

93. RobertsTM

KoborMS

Bastin-ShanowerSA

IiM

HorteSA

2006

Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4.

Mol Biol Cell

17

539

548

94. RobertsTM

ZaidiIW

VaisicaJA

PeterM

BrownGW

2008

Regulation of rtt107 recruitment to stalled DNA replication forks by the cullin rtt101 and the rtt109 acetyltransferase.

Mol Biol Cell

19

171

180

95. ZappullaDC

MaharajAS

ConnellyJJ

JockuschRA

SternglanzR

2006

Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs.

BMC Mol Biol

7

40

96. ScholesDT

BanerjeeM

BowenB

CurcioMJ

2001

Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance.

Genetics

159

1449

1465

97. MunozIM

RouseJ

2009

Control of histone methylation and genome stability by PTIP.

EMBO Rep

10

239

245

98. WangX

TakenakaK

TakedaS

2010

PTIP promotes DNA double-strand break repair through homologous recombination.

Genes Cells

99. CamHP

NomaK

EbinaH

LevinHL

GrewalSI

2008

Host genome surveillance for retrotransposons by transposon-derived proteins.

Nature

451

431

436

100. HeichingerC

PenkettCJ

BahlerJ

NurseP

2006

Genome-wide characterization of fission yeast DNA replication origins.

EMBO J

25

5171

5179

101. BuhlerM

GasserSM

2009

Silent chromatin at the middle and ends: lessons from yeasts.

EMBO J

28

2149

2161

102. HuangY

2002

Transcriptional silencing in Saccharomyces cerevisae and Schizosaccharomyces pombe.

Nucleic Acids Res

30

1465

1482

103. Fernandez-CapetilloO

MahadevaiahSK

CelesteA

RomanienkoPJ

Camerini-OteroRD

2003

H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis.

Dev Cell

4

497

508

104. CabreroJ

TeruelM

CarmonaFD

CamachoJP

2007

Histone H2AX phosphorylation is associated with most meiotic events in grasshopper.

Cytogenet Genome Res

116

311

315

105. BählerJ

WuJ

LongtineM

ShahN

McKenzieA

1998

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.

Yeast

14

943

951

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2010 Číslo 7
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#