New RNA playgrounds : non-coding RNAs and RNA-binding proteins control cellular processes Kedde, M.
19
0
0
Hele tekst
(2) Chapter 4:. Telomerase-independent regulation of ATR by human telomerase RNA J Biol Chem. 2006 Dec 29;281(52):40503-14 DOI 10.1074/jbc.M607676200. 65.
(3) Chapter 4. Telomerase-independent Regulation of ATR by Human Telomerase RNA* S. Received for publication, August 11, 2006, and in revised form, November 6, 2006 Published, JBC Papers in Press, November 10, 2006, DOI 10.1074/jbc.M607676200. Martijn Kedde, Carlos le Sage, Anja Duursma, Eitan Zlotorynski, Bart van Leeuwen, Wouter Nijkamp, Roderick Beijersbergen, and Reuven Agami1 From the Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands The human telomerase RNA (hTR), together with the telomerase reverse transcriptase, hTERT, constitute the core components of telomerase that is essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT is normally restricted to germ cells and certain stem cells, but both are often deregulated during tumorigenesis. Here, we investigated the effects of changes in hTR cellular levels. Surprisingly, while inhibition of hTR expression triggers a rapid, telomerase-independent, growth arrest associated with p53 and CHK1 activation, its increased expression neutralizes activation of these pathways in response to genotoxic stress. These hTR effects are mediated through ATR and are sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses. Furthermore, in response to low UV radiation, which activates ATR, endogenous hTR levels increase irrespective of telomerase status. Thus, we uncovered a novel, telomerase-independent, function of hTR that restrains ATR activity and participates in the recovery of cells from UV radiation.. !?CD 8E=1> C?=1D93 35<<C 81F5 1 <9=9D54 B5@<931D9F5 <965C@1> G85> @B?@171D54 (, 4(20- G8938 9C 4E5 D? D859B 9>129<9DI D? =19> D19> 38B?=?C?=5 5>4C D85 D5<?=5B5C (5<?=5B5C 1B5 C@5 391<9J54 "@B?D59> CDBE3DEB5C D81D @B5C5BF5 D85 9>D57B9DI ?6 D85 5>4C ?6 D85 38B?=?C?=5C 1>4 D85 CD129<9DI ?6 D85 75>?=5 ?G5F5B G9D8 5138 35<< 49F9C9?> D5<?=5B5C 1B5 C8?BD5>54 1>4 ?>35 ?>5 ?B =?B5 5B?45 D? 1 35BD19> 3B9D931< @?9>D D85 C8?BD 5>9>7 9>4E35C 1 " 41=175 3853;@?9>D D85B52I @?C9>7 1 21B B95B D? 3?>D9>E54 35<< 7B?GD8 1>4 D85B56?B5 D? 31>35B
(4) L 5<<C D81D 5C31@5 B5@<931D9F5 C5>5C35>35 2I 9>13D9F1D9>7 3B9D931< 35<< 3I3<5 3853;@?9>D 75>5C CE38 1C @
(5) 3?>D9>E5 D? 5B?45 D859B D5<?=5B5C 5F5>DE1<<I B51389>7 1 @?9>D D81D 9C 31<<54 3B9C9C (85 ?><I G1I D? 5C31@5 3B9C9C 9C D? 5>CEB5 D81D D5<?=5B5C 1B5 =19>D19>54 !?CD 35<<C 4? D89C 2I B513D9F1D9>7 D5<?=5B1C5 2ED. * This work was supported by grants from the Dutch Cancer Society (Koningin Wilhelmina Fonds) (to M. K. and R. A.) and by a EURYI award (to R. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This article was selected as a Paper of the Week. S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S10. 1 To whom correspondence should be addressed: Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands. Tel.: 31-20-512-2079; Fax: 31-20-512-2029; E-mail: r.agami@nki.nl. 66. 1<D5B>1D9F5 =5381>9C=C ( 9>F?<F9>7 B53?=29>1D9?> 1B5 1<C? ?2C5BF54 L (5<?=5B1C5 ?B (&( 9C 1 B92?>E3<5?@B?D59> D81D 3?@95C 1 C8?BD &" D5=@<1D5 9>D? D5<?=5B93 " D85B52I =19>D19>9>7 5E;1BI?D93 38B?=?C?=5 5>4C 1>4 @B5F5>D9>7 B5@<931D9F5 C5>5C 35>35
(6) (5<?=5B1C5 13D9F9DI 9C <?G ?B 12C5>D 9> >?B=1< 8E=1> 35<<C 2ED 9C 8978 4EB9>7 45F5<?@=5>D 9> 35BD19> CD5= 35<<C 75B= 35<<C 1>4 L ?6 8E=1> 31>35BC 1>4 9==?BD1< 9J54 8E=1> 35<< <9>5C . > 613D D5<?=5B1C5 5H@B5C C9?> 9C CE669395>D 6?B 9==?BD1<9J1D9?> ?6 @B9=1BI 8E=1> 35<<C B5>45B9>7 D85= 9>C5>C9D9F5 D? B5@<931D9F5 C5>5C35>35 1>4 3B9C9C > 8E=1>C D85 3?B5 3?=@?>5>DC ?6 D85 D5<?=5B1C5 3?=@<5H 1B5 D85 @B?D59> 31D1<ID93 CE2E>9D 8(&( 1>4 D85 D5<?=5B1C5 &" CE2E>9D 8(& 8(& 9C DB1>C3B9254 2I &" @?<I =5B1C5 1>4 9C
(7) @B?35CC54 D? 75>5B1D5 1 >E3<5?D945<?>7 =1DEB5 DB1>C3B9@D DC C53?>41BI CDBE3DEB5 9C F5BI G5<< 3?> C5BF54 G9D8 D5<?=5B1C5 &"C 6B?= C5F5B1< F5BD52B1D5 C@5395C 9>4931D9>7 1> 9=@?BD1>D B?<5 6?B &" CDBE3DEB5 9> D5<?=5B1C5 6E>3D9?> '5F5B1< @B?D59>C 81F5 255> 45C3B9254 D? 29>4 D? 8(& 1>4 D85C5 1B5 9>F?<F54 9> 8(& CD129<9DI =1DEB1D9?> 133E =E<1D9?> 1>4 6E>3D9?>1< 1CC5=2<I ?6 D85 D5<?=5B1C5 B92?>E3<5 ?@B?D59> 3?=@<5H 5BD19> =ED1D9?>C ?B 45<5D9?>C 9> 8(& <514 D? 1 B1B5 C;9> 1>4 2?>5 =1BB?G 619<EB5 CI>4B?=5 31<<54 4IC;5B1D?C9C 3?>75>9D1 G8938 9C 25<95F54 D? 25 31EC54 2I 45653 D9F5 D5<?=5B5 =19>D5>1>35 9> CD5= 35<<C C 5H@B5CC9?> ?6 8(& 81C 255> 6?E>4 D? 25 5CC5>D91< 6?B D5<?=5B5 =19>D5>1>35 9> 8E=1> 49C51C5 1>4 1<C? 6?B D5<?=5B5 <5>7D8 =19>D5>1>35 9> =?EC5 =?45<C 9D 9C 5H@53D54 D? 25 E@B57E<1D54 9> 31>35B 35<<C >4554 C5F5B1< CDE495C 81F5 C8?G> D81D 8(& E@B57E<1 D9?> 9C 1> 51B<I 5F5>D 9> DE=?B975>5C9C 1>4 D81D 8(& <5F5<C 3?B B5<1D5 25DD5B D? DE=?B 7B145 D81> D5<?=5B1C5 13D9F9DI ?B 8(&( 5H@B5CC9?>
(8) L
(9) )@B57E<1D9?> ?6 8(& G1C 1<C? 6?E>4 D? 25 1> 51B<I 5F5>D 9> =?EC5 =?45<C ?6 DE=?B975>5C9C 5B5 D5<?= 5B1C5 &" <5F5<C 494 >?D @1B1<<5< D85 1=?E>D ?6 D5<?=5B1C5 13D9F9DI 45D53D54
(10)
(11)
(12) (85C5 CDE495C C8?G D81D 5F5> 9> DE=?BC D81D <13;54 D5<?=5B1C5 13D9F9DI D5<?=5B1C5 &" G1C E@B57E<1D54 (85B56?B5 8(& =1I 81F5 6E>3D9?>C D81D 1B5 C5@ 2. The abbreviations used are: ALT, alternative lengthening of telomeres; TERT, telomerase reverse transcriptase; hTR, human telomerase RNA; mTR, mouse telomerase RNA; ATM, ataxia-telangiectasia-mutated protein; ATR, ataxia-telangiectasia and Rad3-related protein; PIKK, phosphatidylinositol 3-kinase-like protein kinase; PBS, phosphate-buffered saline; CMV, cytomegalovirus; TAP, tandem affinity purification; GST, glutathione S-transferase; RT, reverse transcriptase; TRAP, telomeric repeat amplification protocol; TEV, tobacco etch virus; snoRNA, small nucleolar RNA; Gy, gray; RPA, RNase protection assay; siRNA, small interfering RNA; shRNA, short hairpin RNA; IR, ionizing radiation..
(13) ATR regulation by hTR 1B12<5 6B?= 9DC B?<5 9> D5<?=5B1C5 13D9F9DI
(14) ?>DB1CD9>7 D85C5 ?2C5BF1D9?>C 1B5 =935 9> G8938 =?EC5 D5<?=5B1C5 &" =(& G1C 45<5D54 6B?= D85 75B= <9>5 (85C5 =935 1B5 F912<5 6?B C9H 75>5B1D9?>C E>D9< D5<?=5B5C 81F5 3?=@<5D5<I 5B?454
(15) ?G5F5B D85 69BCD 75>5B1D9?> ?6 D85C5 =935 G8938 CD9<< 81F5 <?>7 D5<?=5B5C 81F5 <5CC C;9> DE=?BC D81> G9<4DI@5 =935 6?< <?G9>7 C;9> 385=931< 31B39>?75>5C9C 9>4931D9>7 6?B C?=5 D5<?=5B1C59>45@5>45>D 56653DC ?6 =(&
(16) (89C 1B7E5C D81D =?CD =(& 6E>3D9?> 9C 45@5>45>D ?> D5<?=5B1C5 ?G5F5B 1C 75B= <9>5 75>5 ;>?3;?ED =1I 1<<?G 6?B 3?=@5>C1D9>7 5F5>DC D? ?33EB G89<5 13ED5 9>8929D9?> 9> C?=1D93 35<<C =1I >?D
(17) 9>8929D9?> ?6 D5<?=5B1C5 &" 2I &" 9>D5B65B5>35 9> 31>35B 35<< <9>5C =1I 5H@?C5 9DC D5<?=5B1C5 9>45@5>45>D 6E>3D9?>C (5<?=5B5C 1B5 9>D9=1D5<I <9>;54 G9D8 " 41=175 B5C@?>C5C IC6E3D9?>1< D5<?=5B5C 1B5 B53?7>9J54 1C 41=1754 " 1>4 49B53D<I 1CC?391D5 G9D8 =1>I " 41=175 B5C@?>C5 @B?D59>C
(18) !?B5?F5B D85 =19> 35<<E<1B DB1>C4E35BC ?6 " 41=175 (! 1>4 D? 1 <5CC5B 5HD5>D (& 81F5 255> C8?G> D? @<1I 1> 9=@?BD1>D B?<5 9> D5<?=5B5 8?=5?CD1C9C
(19)
(20) (! 1>4 (& 1B5 @8?C@81D94I<9>?C9D?<
(21) ;9>1C5<9;5 @B?D59> ;9>1C5C $ C D81D 3??B49>1D5 D85 B5@19B 35<< 3I3<5 3853;@?9>D 1>4 1@?@D?D93 B5C@?>C5C D? " 41=175 ?CC ?6 (! 31EC5C D5<?=5B5 4531@@9>7 1>4 C8?BD5>9>7 9> 5F5BI ?B71>9C= 9>F5CD9 71D54 D8EC 61B 1>4 (& 1 D5<?=5B93 " 29>49>7 @B?D59> 81C 255> C8?G> D? 29>4 1>4 9>8929D (! 13D9F1D9?>
(22) $B?2 9>7 1 D5<?=5B93 6E>3D9?> 6?B (& 9> =1==1<91> 35<<C 81C >?D 255> @?CC92<5 C9>35 (& 9C 5CC5>D91< 6?B 35<< F9129<9DI >D5B 5CD9>7<I 1 B535>D B5@?BD 81C C8?G> D81D 9> 0 !(#-.1(1 (& 9C B5AE9B54 6?B =19>D5>1>35 ?6 D5<?=5B93 "
(23) (8EC $ C 6E>3D9?> 9> D5<?=5B5 8?=5?CD1C9C 1>4 D5<?=5B5C G9D8 D859B D5<?=5B51CC?391D54 613D?BC 9>6<E5>35 $ C 13D9F9DI (5<?=5B1C5 1>4 D5<?=5B5C 1B5 1DDB13D9F5 D1B75DC 6?B 31>35B D85B1@95C 1>4 81F5 255> 5HD5>C9F5<I 5H@<?B54 D? D89C 5>4 >8929D9?> ?6 D5<?=5B1C5 2I 1>D9C5>C5 CDB1D5795C ?B 1 4?=9>1>D >571D9F5 8(&( @B?D59> <514C D? D85 5H@53D54 D5<?=5B5 C8?BD 5>9>7 1<D8?E78 D85 7B?GD8 9>8929D9?> 9>4E354 2I D89C =5381 >9C= B5AE9B5C 1 <?>7 <17 @5B9?4 4E5 D? D85 >E=25B ?6 35<< 49F9 C9?>C B5AE9B54 6?B D5<?=5B5C D? 253?=5 CE2CD1>D91<<I C8?BD5>54 D? 9>4E35 7B?GD8 1BB5CD &535>D<I C?=5 CDE495C 81F5 B5@?BD54 B1@94 3ID?D?H93 B5C@?>C5C ?6 31>35B 35<< <9>5C 9> B5C@?>C5 D? <?G <5F5<C ?6 8(&( 1>4 8(& >?F5< D5<?=5B5 9>45@5>45>D 7B?GD8 9>8929D?BI B5C@?>C5 @1D8G1I G1C @B? @?C54 5B5 G5 9>F5CD971D54 D85 56653DC 5H5BD54 2I 8(& ?> D85 35<<E<1B 7B?GD8 1>4 3853;@?9>D 3?>DB?<C 1>4 E>3?F5B54 1 >?F5< D5<?=5B1C59>45@5>45>D 6E>3D9?> ?6 8(& D? 3?E>D5B 21<1>35 D85 13D9F9DI ?6 5>4?75>?EC (&. EXPERIMENTAL PROCEDURES 2$0( *1 ,# ,2(!-#($1M)* B1491D9?> G1C @5B6?B=54 G9D8 1 'DB1D1<9>;5B 'DB1D175>5 1>4 9?>9J9>7 9BB1491D9?> G1C @5B6?B=54 G9D8 1 9
(24) C C?EB35 $B9?B D? )* B1491 D9?> =549E= G1C B5C5BF54 1>4 35<<C G5B5 G1C854 G9D8 @8?C @81D5 2E665B54 C1<9>5 $' >D92?495C EC54 9> D89C CDE4I G5B5 49B53D54 1719>CD 34; @
(25) # '1>D1 BEJ 9?D538 >?<?7I @8?C@8?'5B @
(26) @8?C@8?'5B
(27) 8; @8?C @8?( 8; 5<< '97>1<9>7 ! '97=1 , '5B
(28) )@CD1D5 9?D538>?<?7I 1>4 (& 12 231=. -,1203"21M 8(& ;>?3;4?G> 3?>CDBE3DC G5B5 3<?>54 9> @'E@5B @&5DB?'E@5B@&' I7B? 1>4 @&'$ (85 C5AE5>35C EC54 G5B5 1C 6?<<?GC 8(&;4 ((( ( 8(&;4 ((((( 8(&;4
(29) ((((( (85 !*8(& 5H@B5CC9?> 3?>CDBE3D G1C 3<?>54 2I $& 9> @3"
(30) F53D?B >F9DB?75> D? 3?>D19> >E3<5?D945C 4?G>CDB51= ?6 D85 6E<< <5>7D8 8(& 6?B @B?@5B @B?35CC9>7 2?H C>?&" 3?> CDBE3DC G5B5 1<C? 3<?>54 9>D? D89C F53D?B @&'@
(31) ;4 1>4 @&'&2 G5B5 45C3B9254 @B5F9?EC<I (&;4 C5AE5>35C EC54 G5B5 1C 6?<<?GC (((( ( (( G5 EC54 1 3?=29>1D9?> ?6 D85 DG? 3?>CDBE3DC 3<?>54 9> @'E@5B +5 3<?>54 D85 (& 3" 6B?= D85 @ <17(& 1 ;9>4 796D 6B?= $B?65CC?B 'D5F5 13;C?> F53D?B 9>D? D85 @.?=5 " F53D?B 5<<J?=5 4?G>CDB51= ?6 D85 ($ D17 (85 '(@
(32) 1=9>? 1394C L. 3?>CDBE3D G1C 3<?>54 2I $& 9>D? @, " =B14 @(& $ 1>4 3I 3<?@89<9> 3?>CDBE3DC 6?B D85 &"1C5 @B?D53D9?> 1CC1I &$ G5B5 6B?= =29?> 1>4 D85 8(& &$ F53D?B G1C 3<?>54 2I $& 9>D? @(& =29?> 6B?= >E3<5?D945C L
(33) 9> B5F5BC5 ?B95>D1D9?> $** 3*230$ $20-4(0 * 0 ,1#3"2(-, ,# 0 ,1%$"2(-,M<< 35<<C 45C3B9254 85B5 G5B5 3E<DEB54 9> E<2533?NC =?496954 17<5NC =549E= CE@@<5=5>D54 G9D8 851D9>13D9F1D54 65D1< 31<6 C5BE= 9> # 1D
(34) K 35<<C G5B5 DB1>C4E354 1D @?@ E<1D9?> 4?E2<9>7
(35) (85 75>5B1D9?> ?6 D5BD 35<<C 81C 255> 45C3B9254 @B5F9?EC<I ! 35<<C G5B5 1 ;9>4 796D 6B?= $B?65CC?B 1DC85F1 5B5= ! ) #' 1>4 (
(36) 35<< <9>5C 5H@B5CC9>7 D85 53?DB?@93 B535@D?B G5B5 9>653D54 G9D8 53?DB?@93 B5DB?F9B1< CE@5B>1D1>DC 1C 45C3B9254 @B5F9?EC<I D? 75>5B1D5 @?<I3<?>1< @??<C ?6 35<<C H35@D 6?B ;9>1C5 1CC1IC DB1>C653D9?> G1C 4?>5 2I 5<53DB?@?B1D9?> 1C 45C3B9254 @B5F9?EC<I ?B ;9>1C5 1CC1IC
(37) ) #' 1>4 ! 35<<C G5B5 DB1>C95>D<I DB1>C653D54 EC9>7 31<39E= @8?C@81D5 @B539@9D1D9?> $12$0,
(38) *-22(,& $** 7"*$ 0-%(*$ , *71(1 ,# -+.$2(2(4$ 0-52' 11 71M?B +5CD5B> 2<?D 1>1<IC9C G8?<535<< 5HDB13DC G5B5 @B5@1B54 1>4 C5@1B1D54 ?> ''$ 75<C 1>4 (& 9==E>?2<?DC G5B5 C5@1B1D54 ?> ''$ 75<C 1>4 DB1>C 65BB54 D? ==?29<?>$ =5=2B1>5C !9<9@?B5 +5CD5B> 2<?DC G5B5 45F5<?@54 G9D8 'E@5BC97>1< $95B35 1>4 45>C9D?=5DB93 AE1>D9D1D9?> ?6 +5CD5B> 2<?DC G1C @5B6?B=54 G9D8 941
(39) C?6DG1B5 &1ID5; '856695<4 ) 5<< 3I3<5 @B?69<5 1>1<IC9C G1C @5B6?B=54 1C 45C3B9254 256?B5 2I EEBC=1 $2 * ?B 3?= @5D9D9F5 7B?GD8 1CC1IC 35<<C G5B5 9>653D54 G9D8 @&'$8(&;4 ?B @&'$ B5DB?F9BEC 1>4 1<<?G54 D? B53?F5B 6?B 41IC D85 9>9D91< @5B35>D175 ?6 $@?C9D9F5 35<<C F1B954 25DG55> 1>4 (85 35<<C G5B5 1>1<IJ54 2I 6<?G 3ID?=5DBI G9D8 D85 5<<%E5CD @B?7B1= 9?C395>35 $*-+$0 1$ "2(4(27 11 71 3 ,2(2 2(4$ ,# 1$ 0-2$"2(-, 11 71M! 5HDB13DC G5B5 1CC1I54 6?B D5<?=5B1C5 13D9F9DI EC9>7 1 $&21C54 D5<?=5B93 B5@51D 1=@<9 6931D9?> @B?D?3?< (&$ 1CC1I
(40) ?B &($& 3" G1C DB1>C3B9254 EC9>7 'E@5B'& $( >F9DB?75> G9D8 B1>4?= 85H1=5BC 6?<<?G9>7 D85 =1>E613DEB5BNC 9>CDBE3D9?>C %E1>D9D1 D9F5 $& G1C @5B6?B=54 G9D8 1 '-& B55> =1CD5B =9H @@<954 9?CICD5=C 1>4 D85 C1=@<5C G5B5 1=@<96954 1>4 1>1 <IJ54 2I 1> @B9C= C5AE5>35 45D53D9?> CICD5= @@<954 9?CICD5=C $B9=5BC 6?B #' ?<97?145>I<1D5. 67.
(41) Chapter 4 CI>D85D1C5 G5B5 45C3B9254 256?B5 1>4 2 F1<E5C G5B5 >?B =1<9J54 6?B 13D9> &$C G5B5 @5B6?B=54 EC9>7 D85 I2'@554 &$ 1>4 !, C3B9@D ;9DC 6B?= =29?> 133?B49>7 D? =1>E613 DEB5BNC 9>CDBE3D9?>C +5 EC54 L 7 ?6 &" @5B B513D9?> , 4(20- DB1>C3B9@D9?> ?6 @(& 8(& I95<454 1> &" ?6
(42) >E3<5 ?D945C >E3<5?D945C ?6 G8938 1B5 3?=@<5=5>D1BI D? 8(& ++3,-.0$"(.(2 2(-, (, 1$ 11 71M(? 45D5B=9>5 (& 13D9F9DI ($(& G1C 9==E>?@B539@9D1D54 6B?= 5HDB13DC ?6 DB1>C653D54
(43) ! ?B ) #' 35<<C EC9>7 B1229D 7 '5@81B?C5 '97=1 $B9?B D? 9==E>?@B539@9D1D9?> 35<<C G5B5 )* 9BB1491D54 G9D8 59D85B ?B = ?B =?3; DB51D54 514C G5B5 G1C854 D8B55 D9=5C G9D8 <IC9C 2E665B =! "1< =! 5@5C @ "?>945D $ (G55> =! "1#* =! 7<I35B?@8?C@81D5 1>4 @B?D51C5 9>8929D?B =9HDEB5 &?385 @@<954 '395>35 1>4 (& G1C 3<51F54 6B?= D85 2514C 2I 1449D9?> ?6 B53?=29>1>D (* @B?D51C5 >F9DB? 75> 6?B 8 1D K 133?B49>7 D? =1>E613DEB5BNC 9>CDBE3D9?>C (85 CE2CDB1D5 '(@
(44) L. 2?E>4 D? 7<ED1D89?>5 2514C =5BC81= 9?C395>35C G1C G1C854 D8B55 D9=5C G9D8 ;9>1C5 2E665B =! $' @ =! "1< =! !7< . =! !>< =! 49D89?D8B59D?< =! "1#* 1>4 =! 7<I35B?@8?C@81D5 &513D9?>C G5B5 31BB954 ?ED 9> 1 < F?< E=5 ?6 ;9>1C5 2E665B G9D8 3<51F54 (& 7 ?6 '(@
(45) L. 9 ?6 /
(46) $0($ =9==?< =5BC81= 9? C395>35C 1D
(47) K 6?B =9> > C1=@<5C G9D8 &"1C5 7 G1C 14454 D? D85 ;9>1C5 B513D9?> @B9?B D? D85 1449D9?> ?6 D85 CE2CDB1D5 G5 14454 7 ?6 (, 4(20- DB1>C3B9254 8(& ?B C>?&" D? D85 9>4931D54 C1=@<5C G8938 G1C @B?4E354 6B?= <9>51B9J54 !*8(&C>?&" @<1C=94 G9D8 1 !, C3B9@D (, 4(20- DB1>C3B9@D9?> ;9D =29?> 133?B49>7 D? =1>E613DEB5BNC 9>CDBE3D9?>C (85 2514C G9D8 '(@
(48) L. G5B5 D85> 5HD5>C9F5<I G1C854 1>4 CE2:53D54 D? ''$ 1>4 D85 75< G1C 4B954 CD19>54 G9D8 ??=1CC95 <E5 1>4 5H@?C54 D? 1 @8?C@8? 9=175B C3B55> 6?B AE1>D9D1D9?> ?> 1 1CB5145B
(49) E:9 G9D8 941
(50) C?6DG1B5 &1ID5; '856695<4 ) ++3,-%*3-0$1"$,"$ (2-2(" ,207 ,# 0 &(*$ (2$ 11 71M ! 35<<C G5B5 DB1>C653D54 2I 5<53DB?@?B1D9?> 5669 395>3I G9D8 D85 9>4931D54 3?>CDBE3DC 5<<C G5B5 G1C854 G9D8 $' 69H54 @5B=5129<9J54 9> 6?B=1<458I45 1>4 (B9D?> , 1>4 G1C854 G9D8 $' 3?>D19>9>7 C1@?>9> '<945C G5B5 2<?3;54 G9D8 >?B=1< 7?1D C5BE= 9> $' G9D8 C1@?>9> 5<<C G5B5 CD19>54 G9D8 1>D92?4I 49B53D54 1719>CD @8?C@8?BI<1D54 , 6<E?B5C359> 9C?D89?3I1>1D53?>:E71D54 7?1D 1>D9=?EC5 1>D92?495C G5B5 EC54 1C C53?>41BI 1>D92?495C =175C G5B5 B53?B454 G9D8 1 5931 (' '$ #' 5931 !93B?CICD5=C 5945<25B7 5B=1>I 3?>6?31< CICD5= ?B 3?E>D9>7 =9D?D93 5>DBI 16D5B 8 DB1>C653D54 ! 35<<C G5B5 DB51D54 G9D8 I 9?>9J9>7 B1491D9?> & ?B =?3; DB51D54 1>4 9>3E21D54 G9D8 >?3?41J?<5 7=< 6?B 8 5<<C G5B5 69H54 G9D8
(51) 6?B=1<458I45 @5B=5129<9J54 6?B =9> G9D8 (B9D?> , '97=1 1>4 CD19>54 G9D8 ?538CD !9D?D93 35<<C G5B5 C3?B54 4?E2<5 2<9>4 G9D8 1 .59CC &' =93B?C3?@5 ?B 5138 C1=@<5 35<<C G5B5 3?E>D54 ?B 6B179<5 C9D5 1CC1IC 35<<C G5B5 7B?G> ?> 3?F5BC<9@C 1>4 3?==?> 6B179<5 C9D5C G5B5 9>4E354 2I 7B?G9>7 D85 35<<C 9> ! =549E= 9> D85 @B5C5>35 ?6 ! 1@89493?<9> 1>4 5D81>?< 6?B 8 @B9?B D? D85 69H1D9?> ?6 38B?=?C?=5C 2I CD1>41B4 @B?354EB5C =175C G5B5 ?2D19>54 G9D8 1 .59CC H9?F5BD (* 9>F5BD54 =93B?C3?@5. 68. 3?>DB?<<54 2I '=1BD1@DEB5 C?6DG1B5 ?B 5138 C1=@<5 38B?=?C?=5C G5B5 3?E>D54 4?E2<5 2<9>4. RESULTS ,'(!(2(-, -% ' 6.0$11(-, ,#3"$1 .(# $**3* 0 0-52' 00$12M(? 9>8929D 8(& 5H@B5CC9?> G5 45C97>54 C8&" 3?>CDBE3DC D? D1B75D C5AE5>35C 9> 49665B5>D B579?>C ?6 D85 8(& =?<53E<5 +5 19=54 ?EB 3?>CDBE3DC D? D8B55 49CD9>3D C9>7<5 CDB1>454 <??@C B579?>C ?6 D85 8(& =?<53E<5 1C @B5493D54 2I 9DC F5BI CDB?>7 1>4 3?>C5BF54 C53?>41BI CDBE3DEB5 97 (? 1CC5CC D85 5669313I ?6 9>8929D9?> ?6 8(& 5H@B5CC9?> G5 DB1>C 653D54 ! 2B51CD 31B39>?=1 35<<C G9D8 D85 C8&" 3?>CDBE3DC ?B 1 F53D?B 3?>DB?< +5 EC54 &$ G9D8 &" @B?25C @1BD91<<I 3?=@<5=5>D1BI D? 8(& ?B 3I3<?@89<9> 3?>DB?< D? 1CC5CC 8(& &" <5F5<C 9> 35<< 5HDB13DC 97
(52) %E1>D9D1D9F5 1>1<IC9C B5F51<54 D81D 8(&;4 1>4 8(&;4 B54E354 D85 <5F5<C ?6 5>4?75>?EC 8(& D? 1>4 B5C@53D9F5<I G85B51C >? 8(& B54E3D9?> G1C C55> 2I 8(&;4
(53) ) #' ?CD5?C1B3?=1 35<<C G8938 <13; 5H@B5CC9?> ?6 8(& 1>4 8(&( 2ED =19>D19> D859B D5<?=5B5C 2I ( G5B5 EC54 1C 1 3?>DB?< >4554 >? 8(& 5H@B5CC9?> G1C ?2C5BF54 9> ) #' 35<<C G8938 3?E<4 25 B55H@B5CC54 D8B?E78 DB1>C653D9?> ?6 1 !*8(& @<1C=94 97.
(54) +5 1<C? F5B96954 D85 5HD5>D ?6 8(& ;>?3;4?G> 9> >E3<51B 1>4 3ID?@<1C=93 5HDB13DC D? 5H3<E45 ;>?3;4?G> ?6 8(& ?><I 9> 3ID?@<1C=93 6B13D9?>C CE@@<5=5>D1< 97C ' 1>4 ' +5 6?E>4 D81D 8(& B5C945C =19><I 9> D85 >E3<5EC 1>4 E@?> 5H@B5CC9?> ?6 8(&;4 8(& <5F5<C 9> 2?D8 >E3<51B 1>4 3ID?@<1C=93 6B13D9?>C G5B5 4B1=1D931<<I 453B51C54 (? 1CC5CC D85 56653DC ?6 9>8929D9?> ?6 8(& ?> D5<?=5B1C5 13D9F9DI G5 @5B6?B=54 1 (&$ 1CC1I G9D8 5HDB13DC ?6 ! 35<<C DB1>C653D54 G9D8 D85 8(&;4 3?>CDBE3DC ?B 3?>DB?< @<1C=94 C 5H@53D54 G5 6?E>4 1<=?CD >? D5<?=5B1C5 13D9F9DI 9> 8(&;4 DB1>C653D54 35<<C 1>4 8(&;4 71F5 1 =?4 5B1D5 B54E3D9?> G85B51C 8(&;4
(55) 494 >?D 9>8929D D5<?=5B1C5 13D9F9DI (? 9>F5CD971D5 D85 56653D ?6 8(& ;>?3;4?G> ?> 35<< 7B?GD8 G5 DB1>C653D54 ! 35<<C G9D8 D85 D8B55 8(&;4 3?>CDBE3DC 1>4 1>1<IJ54 D85= 41IC <1D5B 2I 6<?G 3ID?=5DBI 'EB@B9C9>7<I D85 9>DB?4E3D9?> ?6 8(&;4 C8&" F53D?B 9>4E354 1 35<< 3I3<5 1BB5CD 1D 2?D8 1>4 97 G85B51C D85 8(&;4 1BB5CD G1C B54E354 1>4 8(&;4
(56) 814 >? 56653D 41D1 >?D C8?G> (? F5B96I D89C B5CE<D G5 D5CD54 1449D9?>1< 8(&D1B75D9>7 C9&"C G9D8 &$ 1>4 6<?G 3ID?=5DB93 1>1<IC9C 9> ! 35<<C CE@@<5 =5>D1< 97C '
(57) 1>4 ' (B1>C653D9?> ?6 D85C5 C9&"C CE@ @B5CC54 D85 5H@B5CC9?> ?6 8(& 1>4 5<939D54 1 3?=@1B12<5 B1@94 7B?GD8 1BB5CD 1C 8(&;4 ?>C9CD5>D G9D8 D85C5 B5CE<DC D85 <?>7 D5B= CEBF9F1< ?6 8(&;4 DB1>C653D54 ! 35<<C 97 1>4 F9B1<<I DB1>C4E354 8(&;4 ! 35<<C 97 G1C =1B; 54<I 9=@19B54 (85 ?2C5BF54 9>8929D9?> ?6 @B?<965B1D9?> 2I 8(&;4 G1C >?D 1 3?>C5AE5>35 ?6 >?>B5<5F1>D D?H939DI 1C D89C F53D?B 9>4E354 >? 9>D5B65B?> B5C@?>C5 97 EBD85B=?B5 >? 1>D9@B?<965B1D9F5 B5C@?>C5 G1C C55> 9> ) #' 35<<C 1C D85C5 5H@B5CC >? 8(& 97
(58) ?D8 2I 6<?G 3ID?=5DBI 1C G5<< 1C 2I 3?=@5D9D9F5 7B?GD8 1CC1IC 8(&;4 G1C >?D D?H93 D? ) #' 35<<C 2ED G1C 8978<I D?H93 9> ! 35<<C 97 1>4 8(&;4 9>8929D54 7B?GD8 ?6 ! 35<<C D? 9>D5B=5491D5 <5F5<C B56<53D9>7 9DC 31@139DI D? ;>?3;4?G> 8(& (85C5 B5CE<DC 1B5 6EBD85B CDB5>7D85>54 2I D85 EC5 ?6 1449D9?>1< C9&" B5175>DC D1B75D9>7 8(& CE@@<5=5>D1< 97C ' 1>4 '
(59) (89C C8?GC D81D.
(60) ATR regulation by hTR. FIGURE 1. Reduction of telomerase activity and rapid growth inhibition by reduction of hTR expression. A, schematic representation of the hTR RNA. Boxed regions correspond to targeted sequences by shRNAs 1, 2, and 3, and the template sequence is shown in a closed box. B, RPA was used to detect the levels of hTR and the control cyclophilin. The full-length probes and protected fragments are indicated, and 10% of the input probe was loaded on the gel. Quantification was performed by densitometry. C, a TRAP assay was performed to detect telomerase activity in extracts of MCF7 cells transfected with the indicated constructs. D, MCF7 cells were transfected with hTRkd#1 or vector constructs and subjected to flow cytometric analysis. The percentages of cells in G1, S, and G2 phases are shown. S.D. is from three independent experiments. E, a colony growth assay of MCF7 cells transfected with the hTR knockdown constructs or a vector control. Cells were selected with hygromycin for 10 days and stained with Coomassie Blue. F, colony growth assay of virally transduced MCF7 cells. G, MCF7 cells were transfected with indicated constructs, and RNA was extracted after 3 days. OAS1 mRNA levels were measured by quantitative RT-PCR, shown relative to -actin mRNA. S.D. is from three independent experiments. H, flow cytometry is the same as in D only that U2OS cells were used. I, competitive growth assay the U2OS and MCF7 cells were transduced with indicated pRS-GFP constructs. Fluorescence was monitored by flow cytometry at the indicated timepoints after transduction. J, competitive growth assay performed on transduced primary BJ fibroblasts as in I. ctr, control.. D85 7B?GD8 9>8929D9?> DB9775B54 2I D85 ;>?3;4?G> ?6 8(& 9> ! 35<<C 9C 45@5>45>D ?> D85 <5F5< ?6 ;>?3;4?G> ?6 8(& (85 C1=5 1>D9@B?<965B1D9F5 56653D ?6 8(&;4 G1C 1<C? ?2D19>54 9> ?D85B 8E=1> 35<< <9>5C CE38 1C 11( 9==?BD1<9J54 ;5B1D9>? 3ID5C 5 1 35BF931< 31B39>?=1 35<<C 1>4 ( =1==1BI 31B 39>?=1 35<<C CE@@<5=5>D1< 97 ' <D?75D85B D85C5 B5CE<DC C8?G D81D 9>8929D9?> ?6 8(& 5H@B5CC9?> 2I C8&" 5<939DC 1 B1@94 1>D9@B?<965B1D9F5 B5C@?>C5 9> 8E=1> 35<<C E5 D? D85 3?>D9>E?EC B1@94 @B?<965B1D9?> ?6 31>35B 35<< <9>5C 1>4 5=5B75>35 ?6 3B9D931<<I C8?BD D5<?=5B5C D85 9>8929D9?> ?6 8(& 5H@B5CC9?> =1I 9>4E35 1 CDB5CC B5C@?>C5 (? 5H1=9>5 D89C G5 CDE4954 8(& 9>8929D9?> 9> I?E>7 @B9=1BI 8E=1> 35<<C @1C C175
(61) D81D 5H@B5CC 8(& 1>4 3?>D19> CE669395>D<I <?>7 D5<?=5B5C D? =19>D19> @B?<965B1D9?> 6?B
(62) L 1449D9?>1< @1C. C175C <C? 9> D85C5 35<<C CE@@B5CC9?> ?6 8(& 9>8929D54 35<< 7B?GD8 45@5>49>7 ?> D85 <5F5< ?6 8(& ;>?3;4?G> 97 <D?75D85B ?EB B5CE<DC 9>4931D5 D81D 9>8929D9?> ?6 8(& 5H@B5C C9?> 9>4E35C 1 35<< 3I3<5 1BB5CD G8938 9C >?D 31EC54 2I 3B9D931<<I C8?BD D5<?=5B5C $** 7"*$ 00$12 ,#3"$# !7 ,'(!(2(-, -% ' 6.0$11(-, $/3(0$1 . ,# M(? 5<E3941D5 D85 B5C@?>C5 ?6 35<<C D? B54E354 8(& <5F5<C G5 =?>9D?B54 @
(63) <5F5<C 1 DE=?B CE@@B5C C?B 1>4 1 =1:?B DB1>C4E35B ?6 35<< 3I3<5 1BB5CD 9> B5C@?>C5 D? ?>3?75>93 1>4 75>?D?H93 CDB5CC5C +5 DB1>C653D54 ! 35<<C G9D8 @
(64) ;4 1>4 8(&;4 ?B 3?>DB?< 3?>CDBE3DC 1>4 6?E>4 @
(65) @B?D59> <5F5<C D? 25 D8B55 D9=5C 89785B 9> D85 8(&;4 DB1>C 653D54 35<<C 3?=@1B54 G9D8 3?>DB?<C 97 (89C CE775CDC D81D D85 7B?GD8 1BB5CD ?2C5BF54 9> 8(&;4 35<<C 9>F?<F5C @
(66) 13D9F1. 69.
(67) Chapter 4. FIGURE 1—continued. D9?> (? D5CD D89C 49B53D<I G5 @5B6?B=54 3?=@5D9D9F5 7B?GD8 1CC1IC EC9>7 !@
(68) ;4 1>4 ! 3?>DB?< 35<<C D81D G5B5 F9B1<<I DB1>C4E354 G9D8 D85 $8(&;4 ?B $ 3?>DB?< F53 D?B 97
(69) C8?GC D81D <?CC ?6 @
(70) 5H@B5CC9?> G8938 3?=@<5D5<I 12B?71D5C 1 " 41=175 B5C@?>C5 ?><I @1BD91<<I B5C3E54 D85 1BB5CD 9>4E354 2I <?CC ?6 8(& C9=9<1B B5CE<D G1C ?2D19>54 9> (
(71) @B9=1BI 692B?2<1CDC CE@@<5=5>D1< 97 ' 3D9F1D9?> ?6 @
(72) 31> 25 1 B5CE<D ?6 13D9F1D9?> ?6 D85 ;9>1C5C (! 1>4 (& G8938 9> DEB> 13D9F1D5 1>4 B5C@53D9F5<I (? 5H1=9>5 G85D85B D85 35<< 3I3<5 1BB5CD. 70. 9>4E354 2I D85 <?CC ?6 8(& 45@5>4C ?> D85 3?=29>1D9?> ?6 @
(73) 1>4 G5 @5B6?B=54 1 $ 3?=@5D9D9?> 1CC1I G9D8 1>4 @
(74) ;4 35<<C 9> D85 @B5C5>35 ?6 D85 9>8929D?B )" ?B F5893<5 1C 3?>DB?< 97 C8?GC D81D 8(&;4 9>4E354 D?H939DI G1C 1<=?CD 3?=@<5D5<I 12B?71D54 G85> 2?D8 @
(75) 5H@B5CC9?> 1>4 13D9F9DI G5B5 9>8929D54 '9=9<1B B5CE<DC G5B5 ?2D19>54 9> ! 35<<C 41D1 >?D C8?G> (85C5 B5CE<DC 9>4931D5 D81D <?CC ?6 8(& 13D9F1D5C 2?D8 @
(76) 1>4 D? 5<939D 1 B1@94 35<< 3I3<5 1BB5CD 9>4931D9>7 D85 9>F?<F5=5>D ?6 (& 9> D89C @B?35CC.
(77) ATR regulation by hTR
(78)
(79) D9=5 (? 5H3<E45 ?66 D1B75D 56653DC ?6 8(& ;>?3;4?G> G5 D5CD54 1449D9?>1< C9&"C D81D 9>8929D 8(& 5H@B5CC9?> +5 6?E>4 1<C? D8?C5 D? 5<939D 1 B1@94 35<< 3I3<5 1BB5CD 1>4 D? DB9775B '
(80) 1>4 @
(81) ' @8?C@8?BI<1D9?> 1>4 @
(82) CD129<9J1D9?> CE@@<5=5>D1< 97C '
(83) ' 1>4 ' (89C CDB?>7<I CE7 75CDC D81D 9>8929D9?> ?6 8(& 31EC5C D85 B54E3D9?> 9> 35<< @B?<965B1D9?> 31@139DI 1>4 13D9F1D9?> ?6 " 41=175 3853;@?9>DC >D5B5CD9>7 <I 8(& 45@<5D9?> 13D9F1D54 D85 " 41=175 B5C@?>C5 D? 1<=?CD 6E<< 5HD5>D 1C DB51D9>7 8(&;4 35<<C G9D8 )* B5CE<D54 9> ?><I 1 C<978D 13D9F1D9?> ?6 @
(84) 1>4 3?= @1B54 G9D8 3?>DB?< 35<<C
(85) <D?75D85B D85C5 B5CE<DC CE775CD D81D 5>4?75>?EC <5F5<C ?6 kd FIGURE 2. Chk1 and p53 are required for induction of the hTR -mediated cell cycle arrest. A, MCF7 cells were transfected with indicated constructs and subjected to immunoblot analysis to detect p53 and the 8(& 1B5 6E>3D9?>9>7 D? 21<1>35 control CDK4. Band intensity was calculated by densitometry. B, MCF7 cells were transduced with p53kd or (& 13D9F9DI 1 6E>3D9?> D81D 9C control vector, drug-selected, and transduced with pRS-GFP control and hTRkd#1-GFP. Competitive growth D5<?=5B1C59>45@5>45>D assays were performed as described in the legend to Fig. 1I. C, competitive growth assays with BJ cells as in B. (? CDE4I D85 56653DC ?6 8(& ?> Cells were either treated with UCN-01 (10 nM, dashed lines) or with vehicle (dimethyl sulfoxide (DMSO), solid lines). (& ;9>1C5 13D9F9DI 9> =?B5 45D19< G5 EC54 1> (& 3?>CDBE3D G9D8 1 ' ,'(!(21 "2(4(27M#EB B5CE<DC CE775CD 1> 9>F5BC5 ($ D1>45= 1669>9DI @EB96931D9?> D17 3?>D19>9>7 1 (* @B?D5 3?BB5<1D9?> 25DG55> 8(& <5F5<C 1>4 (& 13D9F9DI &54E3D9?> 9> 1C5 3<51F175 C9D5 +5 DB1>C653D54
(86) 35<<C G9D8 ($ 8(& <5F5<C 9>4E35C @
(87) 1>4 DG? =19> CE2CDB1D5C ?6 (& (& ?B ($ 3?>DB?< @EB96954 D85= 6B?= 5HDB13DC 2I 9==E>? (? 5H1=9>5 D89C 6EBD85B G5 1C;54 G85D85B D85 @B5C5>35 ?6 8(& @B539@9D1D9?> EC9>7 7 2514C 97
(88) * ,$1
(89) 1>4 B5<51C54 9>8929DC (& 13D9F9DI (& 9C 13D9F1D54 2I " 41=175 1CC1E<DC EC9>7 (* @B?D51C5 * ,$1 +5 CDE4954 56653DC ?6 8(& ?> CE38 1C )* B1491D9?> D85B52I 9>4E39>7 D85 @8?C@8?BI<1D9?> ?6 (&;9>1C5 13D9F9DI EC9>7 '(3?E@<54 @
(90) 1=9>? 1394C @
(91) ' 1>4 '
(92) B5C@53D9F5<I L (? D5CD D85 B?<5 ?6 L. 1C 9DC CE2CDB1D5 97
(93) C8?GC D81D ?EB @EB96954 (& 8(& 9> D89C @B?35CC G5 EC54 ) #' 35<<C G8938 5H@B5CC >? 8(& 3?E<4 @8?C@8?BI<1D5 @
(94) 1C 5H@53D54 * ,$ "20* +85> 8(& 9C 1>4 53D?@931<<I 5H@B5CC54 8(& 97 =@?BD1>D<I D85 <5F5<C 9>3E21D54 G9D8 (& 1 3<51B 1>4 @?D5>D 453B51C5 9> (& 13D9F9DI ?6 5H@B5CC54 8(& 9> ) #' 35<<C G5B5 <?G5B D81> D85 5>4?75 9C ?2C5BF54 * ,$ ' C 3?>DB?< G5 EC54 1 DBE>31D54 8(& >?EC <5F5<C ?2C5BF54 9> ! 35<<C 1>4 3?E<4 >?D 3?=@<5=5>D &" 5>3?=@1CC9>7 D85 69BCD . >E3<5?D945C ?6 8(& * ,$ ' 6?B D5<?=5B1C5 13D9F9DI 4E5 D? D85 <13; ?6 8(&( 5H@B5CC9?> 9> (89C DBE>31D54 8(& 9>8929D54 (& 13D9F9DI (, 4(20- D? 1 D85C5 35<<C 41D1 >?D C8?G> +5 9BB1491D54 8(& 5H@B5CC9>7 1>4 <5CC5B 5HD5>D D81> D85 6E<<<5>7D8 8(& 9>4931D9>7 6?B D85 C@539 3?>DB?< 35<<C G9D8 )* 1>4 6?<<?G54 D85 @8?C@8?BI<1D9?> ;9>5D 6939DI ?6 D85 8(& 56653D 449D9?>1<<I C9>35 8(& 3?>D19>C 1 93C ?6 '
(95) 1>4 @
(96) ' 9> D9=5 >D5B5CD9>7<I E@?> B1491 2?H G5 D5CD54 B5<1D54 C>?&"C 6?B D9?> D85 @8?C@8?BI<1D9?>C ?6 2?D8 '
(97) 1>4 @
(98) ' G5B5 D859B 129<9DI D? 9>6<E5>35 (& ;9>1C5 13D9F9DI +5 6?E>4 D81D C5F5B5<I 1DD5>E1D54 9> ) #' 35<<C 5H@B5CC9>7 8(& 97
(99) > D85C5 &"C 81B4<I 16653D (& ;9>1C5 13D9F9DI 9>4931D9>7 D81D 1449D9?> @
(100) CD129<9DI G1C >?D 9>3B51C54 E@?> B1491D9?> 9> 8(& D85 9>8929D9?> ?6 (& 2I 8(& 9C C@539693 CE@@<5=5>D1< 97 ' C
(101) 35<<C 5H@B5CC 8(& G5 B51C?>54 D81D 1449D9?> ?6 5H@B5CC9>7 35<<C 1C 3?=@1B54 G9D8 3?>DB?< 35<<C '9>35 ) #' 35<<C 5H@B5CC >59D85B 8(& >?B 8(&( 9D &"1C5 C8?E<4 9>3B51C5 (& 13D9F9DI >4554 G85> &"1C5 G1C C55=C D81D 8(& 9>8929DC (& 9> 1> 8(&( 9>45@5>45>D 14454 D? (& ?B D? (& 1>4 8(& ;9>1C5 13D9F9DI G1C B5<1D9F5<I =1>>5B (? 5H1=9>5 D89C 9CCE5 G5 19=54 1D D5CD9>7 D85 56653DC 9>3B51C54 (85C5 B5CE<DC 9>4931D5 D81D (& ;9>1C5 13D9F9DI 9C ?6 8(& ;>?3;4?G> 9> 1 D5<?=5B1C5 >571D9F5 35<< <9>5 (85B5 9>8929D54 2I 8(& (, 4(20- (? 6EBD85B 5H1=9>5 D85 56653DC ?6 8(& ?> (& ;9>1C5 13D9F9DI 6?B5 G5 EC54 ! 8E=1> 692B?2<1CDC D85C5 35<<C 5<?>71D5 D5<?=5B5C 2I D85 ( =5381>9C= 1>4 5H@B5CC 8(& 2ED >?D G5 @5B6?B=54 ;9>1C5 1CC1IC G9D8 ($(& 9> ) #' 35<<C D81D 8(&( +5 DB1>C653D54 35<<C G9D8 D85 8(&;4 3?> <13; 8(& 5H@B5CC9?> 5<<C G5B5 DB1>C653D54 G9D8 ($(& ?B CDBE3D DB51D54 D85 35<<C G9D8 )* B1491D9?>
(102) = 1>4 =?> ($3?>DB?< @<1C=94 1>4 3?DB1>C653D54 G9D8 !*8(& ?B 9D?B54 @8?C@8?BI<1D9?> ?6 '
(103) 1>4 @
(104) ' EC9>7 9==E 3?>DB?< @<1C=94C (8B55 41IC 16D5B DB1>C653D9?> 35<<C G5B5 >?2<?D 1>1<IC9C +5 6?E>4 D81D B54E3D9?> 9> 5>4?75>?EC 59D85B <56D E>DB51D54 ?B )* 9BB1491D54 D? 13D9F1D5 (& 1>4 CE2 8(& <5F5<C DB9775B54 '
(105) 1>4 @
(106) ' @8?C@8?BI<1D9?> C5AE5>D<I (& G1C @EB96954 1>4 CE2:53D54 D? 1 ;9>1C5 1CC1I 97 1C G5<< 1C @
(107) CD129<9J1D9?> 9>4931D9>7 (& 13D9F1D9?> 97
(108) C8?GC D81D 5H?75>?EC (& 9C 13D9F5 9> =?3; DB51D54 ) #'. 71.
(109) Chapter 4 3?>DB?< DB1>C653D9?> * ,$1 1>4 "5HD G5 @5B6?B=54 1 C9=9<1B 5H@5B 9=5>D EC9>7 ! 35<<C D81D E><9;5 ) #' 35<<C 5H@B5CC 8(& 97
(110) C8?GC (& 13D9F1D9?> E@?> )* B1491D9?> * ,$1 8 ?G5F5B B54E3D9?> 9> 8(& <5F5<C 9>4E354 (& 13D9F9DI 1<B514I 9> D85 E>DB51D54 35<<C * ,$ G8938 G1C >?D 6EBD85B 13D9F1D54 2I )* 1D D89C D9=5 @?9>D 16D5B DB51D=5>D * ,$ C C8?G> 12?F5 (& C97>1<9>7 9C 13D9F1D54 G85> 8(& 9C 45@<5D54 #EB B5CE<DC C? 61B CE775CD D81D D85 56653D ?6 8(& ?> (& 13D9F9DI 9C 49B53D (& 9C >?B=1<<I 13D9F1D54 2I " 41=175 E@?> G8938 9D 13D9 F1D5C @
(111) 1>4 2ED 1<C? @8?C @8?BI<1D5C 89CD?>5 , $8?C@8?BI<1D54 , , 3?<?31<9J5C G9D8 ?D85B 613D?BC CE38 1C @
(112) $ !&. 1>4 '! D? 6?39 =1B;9>7 C9D5C ?6 " 41=175 D85B52I 6139<9D1D9>7 D85 1CC5=2<I ?6 3853;@?9>D 1>4 " B5@19B 613D?BC 'E38 6?39 1B5 1<C? 6?E>4 1D 4IC 6E>3D9?>1< D5<?=5B5C
(113) (? D5CD G85D85B 8(& 45@<5D9?> 13DC 49B53D<I D8B?E78 (& ?B 9>49B53D<I F91 9>4E3D9?> ?6 " 41=175 G5 CD19>54 8(&;4 35<<C 6?B , 6?39 ! 35<<C G5B5 DB1>C653D54 G9D8 8(&;4 ?B F53D?B 3?>DB?< ?B 9BB1 491D54 1C @?C9D9F5 3?>DB?< +89<5 & 75>5B1D54 , 6?39 >? 9>3B51C5 9> 6?39 6?B=1D9?> 12?F5 3?>DB?< G1C ?2C5BF54 9> 35<<C 5H@B5CC9>7 8(&;4 C8&" 8(&;4 C9&" 41D1 >?D C8?G> 97
(114) C9=9<1B B5CE<D G1C ?2D19>54 @B5F9?EC<I G9D8
(115) $. 6?39 9> #, 35<<C (8EC G5 6?E>4 >? 5F945>35 ?6 " 41=175 9> 35<<C 45@<5D54 ?6 8(& CE@@?BD9>7 FIGURE 3. hTR inhibits ATR kinase activity. A, U2OS cells were transfected with the hTRkd or control, irradiated D85 >?D9?> D81D D85 56653DC ?6 8(& ?> after 3 days with 3 J/m2 UV, and harvested at the indicated time points. Whole cell extracts were immuno- (& 1B5 49B53D <D?75D85B ?EB blotted to detect CHK1S317 phosphorylation, p53S15 phosphorylation, p53, and CDK4 as a loading control. B, B5CE<DC C8?G D81D D85 5H@B5CC9?> ?6 GM847 fibroblasts were transfected with hTRkd or control and treated as in A. C, HEK293 cells were transfected with TAP-ctr or TAP-ATR and immunoprecipitated with IgG. TAP-ATR was cleaved with TEV protease, and beads 8(& 9>6<E5>35C (& ;9>1C5 13D9F9DI (B) and supernatant (S) were immunoblotted to detect ATR. WB, Western blot. D, kinase assay performed with (, 4(4- immunoprecipitation and TEV cleaved TAP-ATR from HEK293 cells with GST-p53 (residues 1–101) as a sub 02( * !0-& 2(-, -% 2'$ strate. Cleaved ATR was split, and hTR, RNase, and mock were added prior to kinase reaction. Samples were separated by 10% SDS-PAGE and stained with Coomassie Blue to detect GST-p53 protein, and autoradiogra- '$").-(,2 ,# ,' ,"$# ..$ 0 phy was performed to detect kinase activity. Band intensities were measured by densitometry. E, U2OS cells ,"$ -% 0 &(*$ (2$1 !7 ' were transfected with the indicated constructs and kinase assays were performed as in B. F, MCF7 cells were transfected with the indicated constructs, and kinase assays were performed as in B. G, immunofluorescence 6.0$11(-,M(& 81C 255> C8?G> kd images of MCF7 cells transfected with hTR #1, vector, or control irradiated cells (5 Gy IR), stained with -H2AX D? 25 9>F?<F54 9> D85 B1491D9?>9> antibody. Nuclei are stained with DAPI (blue). Pictures were made with 200 magnification. vec, vector. 4E354 ! @81C5 3853;@?9>D 9> 5E;1BI?D93 35<<C 1C 9D @B5F5>DC 35<<C 1>4 9C C<978D<I 13D9F1D54 G85> 35<<C 1B5 9BB1491D54 * ,$1 =9D?D93 5>DBI ?6 35<<C =19><I 9> D85 <1D5 @81C5 ?6 D85 B5C@?>C5 D? 1>4 ?G5F5B G85> 8(& 9C 3?5H@B5CC54 G9D8 ($(& & 1>4 3??@5B1D5C G9D8 (! 9> D85 51B<I @81C5 ?6 D85 B5C@?>C5 (& 13D9F9DI 9C =1B;54<I 453B51C54 1<=?CD D? D85 <5F5<C ?6 D85 '9>35 ?EB B5CE<DC C8?G D81D 8(& 9>8929DC (& G5 CDE4954. 72.
(116) ATR regulation by hTR. FIGURE 4. hTR partially abrogates the G2/M checkpoint and enhances the expression of fragile sites. A, MCF7 cells were transfected with the indicated constructs irradiated with 5 Gy IR or mock treated 3 days later and incubated with nocodazole. Twenty-our hours later, cells were fixed and stained with Hoechst, and mitotic entry was scored. S.D. from three independent experiments is shown. B, the expression of fragile sites in U2OS cells transfected with the indicated constructs. Pictures were made with 1000 magnification. C, quantification of the expression of fragile sites in U2OS cells as shown in Fig. 4B. The number of fragile sites expressed in 400 chromosomes is shown; data are representative of three independent experiments.. D85 56653DC ?6 8(& 5H@B5CC9?> ?> D85 ! 3853;@?9>D +5 =?>9D?B54 =9D?D93 5>DBI ?6 ! 35<<C 9> B5C@?>C5 D? & 1C 1 =51CEB5 6?B D85 >E=25B ?6 35<<C 12<5 D? 1BB5CD 9> ! +5 DB1>C653D54 ! 35<<C G9D8 !*8(& 1>4 1C 3?>DB?<C (&;4 @
(117) ;4 ?B !*3?>DB?< 3?>CDBE3DC 6?B F1<941D9?> ?6 D85 (&;4 3?>CDBE3DC C55 CE@@<5=5>D1< 97 ' (8B55 41IC <1D5B G5 9BB1 491D54 35<<C G9D8 I & 1>4 DB51D54 D85 35<<C G9D8 >?3?41J?<5 D? 9>8929D @B?7B5CC9?> ?6 D85 35<< 3I3<5 9> =9D?C9C 5<<C G5B5 D85> 69H54 CD19>54 G9D8 ?538CD 1>4 =9D?D93 35<<C G5B5 3?E>D54 97 +89<5 G5 6?E>4 >? 49665B5>35 9> D85 133E=E<1D9?> ?6 =9D?D93 35<<C 9> E>9BB1491D54 3?>DB?<C 8(& ?F5B5H@B5CC9?> (&;4 1>4 @
(118) ;4 C8?G54 1 @1BD91< ?F5BB945 ?6 D85 ! 1BB5CD > 3?>DB1CD 35<<C 5H@B5CC9>7 1 DBE>31D54 6?B= ?6 8(& 8(&. 2581F54 1C 3?>DB?< 35<<C "?D12<I 8(& <5F5<C 1B5 ?><I =?45B 1D5<I 9>3B51C54 9> ! 35<<C E@?> 5H@B5CC9?> ?6 !*8(& 1
(119) 9>3B51C5 CE@@<5=5>D1< 97 ' <D?75D85B D85C5 B5CE<DC 9>4931D5 D81D 1> 9>3B51C5 9> 8(& <5F5<C 9=@19BC (&=5491D54 " 41=175 B5C@?>C5C 9> 1 =1>>5B C9=9<1B D? D8?C5 9>4E354 2I <?CC ?6 (&. (? 6EBD85B 9>F5CD971D5 G85D85B 8(& 5H@B5CC9?> @5BDEB2C (& 6E>3D9?> G5 CDE4954 6B179<5 C9D5C CD129<9DI 9> ) #' 35<<C ?==?> 6B179<5 C9D5C 1B5 C@539693 38B?=?C?=1< <?39 D81D 1@@51B 1C 3?>CDB93D9?>C 71@C ?B 2B51;C ?> =5D1@81C5 38B?=?C?=5C 6B?= 35<<C D81D 81F5 255> 5H@?C54 D? @1BD91< 9>8929D9?> ?6 " B5@<931D9?>
(120) 449D9?> ?6 <?G <5F5<C ?6 D85 " @?<I=5B1C5 9>8929D?B 1@89493?<9> D? 35<<C 9> 3E<DEB5 81C 255> C8?G> D? 9>4E35 6B179<5 C9D5C (& B53?7>9J5C CD1<<54 1>4 3?<<1@C54 " B5@<931D9?> 6?B;C 1>4 13D9F1D5C D859B B5@19B 1>4 D85 B5CD1BD9>7 ?6 B5@<931D9?> B179<5 C9D5C 1B5 25<95F54 D? 25 5H@B5CC54 G85> CD1<<54 6?B;C 5C31@5 D85 (& B5@<931D9?> CEBF59<<1>35 +5 DB1>C653D54 ) #' 35<<C G9D8 !*8(& (&;4 ?B !*3DB< 1>4 DB51D54 D85 35<<C 6?B 8 G9D8 1@89493?<9> 8B?=?C?=5C G5B5 69H54 CD19>54 1>4 4B?@@54 ?>D? C<945C D? 3?E>D 38B?=? C?=5C 1>4 6B179<5 C9D5C )@?> 9>8929D9?> ?6 B5@<931D9?> G5 6?E>4 6B179<5 C9D5C 9>CD129<9DI 9> 3?>DB?< 35<<C G8938 G1C 5H135B21D54 2I 59D85B <?CC ?6 (& ?B 5H@B5CC9?> ?6 8(& 97
(121) %E1>D969 31D9?> ?6 D85 6B5AE5>3I ?6 6B179<5 C9D5C 5H@B5CC9?> C8?G54 1 6?<4 9>3B51C5 9> D85 >E=25B ?6 6B179<5 C9D5C 9> D85 8(& 5H@B5CC. 73.
(122) Chapter 4. FIGURE 5. Up-regulation of hTR levels following UV radiation. A, RPA performed on extracts from BJ cells and BJ cells immortalized with hTERT irradiated with 3 J/m2 UV or 4 Gy ionizing radiation. Cells were harvested after irradiation at the indicated time points, and quantification was performed by densitometry. B, quantification of RPAs on extracts of BJ cells irradiated with either 3 J/m2 UV or 4Gy ionizing radiation. Samples were harvested at indicated time points after irradiation. S.D. is from three independent experiments; band intensities were quantified by densitometry. C, RPA performed on extracts of GM847 cells irradiated with 3 J/m2 UV as in A. D, quantification of RPAs on extracts of the indicated cells irradiated with 3 J/m2 UV. UCN-01 was added to a final concentration of 100 nM 1 h prior to irradiation. S.D. is from three independent experiments. Ctr, control; Vec, vector.. 9>7 35<<C 1C G5<< 1C 9> D85 (&;4 35<<C 97 C =19>D19>9>7 <?G <5F5<C ?6 6B179<5 C9D5 5H@B5CC9?> 1B5 1 @B9=5 6E>3D9?> ?6 (& ?EB B5CE<DC 9>4931D5 D81D 8(& 9>8929DC (& 13D9F9DI <D?75D85B D85C5 B5CE<DC C8?G D81D 8(& 9>8929DC (& 13D9F9DI D? 1 <5F5< CE669395>D D? 9=@19B 49665B5>D (&=5491D54 "41=175 3853;@?9>D B5C@?>C5C 9=@<I9>7 1 >?F5< 6E>3D9?> 6?B 8(& ' .0$&3* 2(-, %-**-5(,& #( 2(-,M+5 >5HD C5D ?ED D? 5<E3941D5 D85 6E>3D9?> ?6 D85 9>8929D?BI 56653D ?6 8(& ?> (& (? D89C 5>4 G5 9>F5CD971D54 G85D85B CD9=E<9 D81D 13D9F1D5 (& 1<C? 9>6<E5>35 D85 5H@B5CC9?> ?6 8(& +5 DB51D54 @B9=1BI 692B?2<1CDC 1>4 35<<C ?F5B5H@B5CC9>7 8(&( ( G9D8
(123) = )* <978D 1>4 5HDB13D54 &" 1D C5F5B1< D9=5 @?9>DC 16D5B B1491D9?> 'E2C5AE5>D<I G5 CE2:53D54 D85C5 &" 5HDB13DC D? &$ G9D8 1> 8(& @B?25 1>4 3I3<?@89<9> ?B $ 1C 3?>DB?<C 97 C8?GC D81D 6?<<?G9>7 )* B1491D9?> 2ED >?D & I 8(& <5F5<C 9>3B51C5 9> @1B5>D 35<<C > ( 35<<C 1C @E2 <9C854 @B5F9?EC<I
(124) 89785B <5F5<C ?6 8(& 1B5 45D53D54 9> E>DB51D54 35<<C 'D9<< 1 3<51B E@B57E<1D9?> ?6 8(& <5F5<C 6?<<?G 9>7 )* DB51D=5>D 9C 45D53D54 97 (85 9>3B51C5 9> 8(&. 74. <5F5< 6?<<?G9>7 )* B1491D9?> G1C >?D 4E5 D? 1 35<< 3I3<5 1BB5CD ?6 D85 35<<C 1C 8(& <5F5<C 494 >?D 9>3B51C5 9> &DB51D54 35<<C 97 1>4
(125) "5HD G5 381B13D5B9J54 D85 B5C@?>C5 ?6 8(& D? )* 9BCD G5 5H1=9>54 45@5>45>35 ?> 8(&( 2I EC9>7 ! 35<<C D81D 5H@B5CC 8(& 2ED >?D 8(&( <C? 9> D85C5 35<<C 8(& G1C E@ B57E<1D54 9> B5C@?>C5 D? )* B1491D9?> 97 9>4931D9>7 D81D D85 E@B57E<1D9?> ?6 8(& 9C 9>45@5>45>D ?6 8(&( '53 ?>4 G5 9>F5CD971D54 G85D85B D85 E@B57E<1D9?> ?6 8(& 9C 45@5>45>D E@?> @
(126) ?B 13D9F9DI 2I EC9>7 @
(127) ;4 35<<C 1>4 35<<C DB51D54 G9D8 )" > D85C5 5H@5B9=5>DC 8(& G1C CD9<< E@B57E<1D54 9> B5C@?>C5 D? )* B1491D9?> G85> 3?=@1B54 G9D8 3?>DB?< D9=5 @?9>DC 9>4931D9>7 D81D @
(128) 1>4 1B5 >?D 9>F?<F54 9> D85 E@B57E<1D9?> ?6 8(& 9> B5C@?>C5 D? )* B1491D9?> 97 ?<<53D9F5<I G5 E>3?F 5B54 D81D 8(& 9C C@5396931<<I E@B57E<1D54 9> B5C@?>C5 D? <?G <5F5<C ?6 )* B1491D9?> (89C E@B57E<1D9?> 9C >?D ?2C5BF54 9> B5C@?>C5 D? 9?>9J9>7 B1491D9?> 1>4 9C 9>45@5>45>D ?6 @
(129) 1>4 8(&(.
(130) ATR regulation by hTR DISCUSSION +5 81F5 945>D96954 1 31EC1D9F5 75>5D93 9>D5B13D9?> 25DG55> D85 8(& 1>4 D85 3853;@?9>D ;9>1C5 (& 3D?@93 5H@B5CC9?> ?6 8(& 9>8929DC (& G89<5 B54E3D9?> 9> 8(& <5F5<C CD9=E<1D5C (& 13D9F9DI (89C 9>D5B13D9?> 9C 9>45@5>45>D ?6 D5<?=5B1C5 13D9F9DI 1>4 D5<?=5B5 <5>7D8 1C 9D G1C ?2C5BF54 9> 35<<C <13;9>7 8(&( 1>4 9> I?E>7 @B9=1BI 8E=1> 35<<C G9D8 D5<?=5B5C <?>7 5>?E78 D? 1<<?G @B?<965B1D9?> 6?B 1D <51CD D?
(131) @1CC175C >D5B5CD9>7<I D85 56653D ?6 8(& ?> (& 13D9F9DI G1C CDB?>7 5>?E78 D? 9>6<E5>35 35<<E<1B @1D8G1IC >8929D9?> ?6 8(& 5H@B5CC9?> 5<939D54 1 @
(132) 45@5>45>D 35<< 3I3<5 1BB5CD 9> D85 12C5>35 ?6 1@@1B5>D " 41=175 G89<5 9>3B51C54 5H@B5C C9?> ?6 8(& 31EC54 45653DC 9> (&45@5>45>D 3853;@?9>DC CE38 1C ?F5BB945 ?6 D85 ! 1BB5CD 9> B5C@?>C5 D? " 41=175 1>4 D85 5>81>354 9>4E3D9?> ?6 6B179<5 C9D5C (85C5 56653DC 1B5 C@539693 C9>35 D85 8(& ;>?3;4?G>C 4? >?D 5<939D 1>I 9>D5B65B?> B5C@?>C5 1>4 @85>?DI@931<<I 45@5>4 ?> D85 5>4?75>?EC 5H@B5CC9?> ?6 8(& 1>4 D85 56653DC G5B5 ?2C5BF54 G9D8 =9<4 <5CC D81> 12?F5 5>4?75>?EC ?F5B5H@B5CC9?> ?6 8(& "?D12<I CE38 1 =9<4 9>3B51C5 9> 8(& 5H@B5CC9?> 9C 1<C? ?2C5BF54 G85> 35<<C 1B5 )* 9BB1491D54 9>4931D9>7 D81D CE38 1> 9>3B51C5 9C CE6 69395>D D? 9=@19B (& 45@5>45>D 3853;@?9>DC 97
(133) > 9=@?BD1>D ?2C5BF1D9?> 9C D81D 2?D8 D85 9>8929D9?> ?6 (& 13D9F 9DI 2I 8(& 1>4 D85 E@B57E<1D9?> ?6 8(& 2I )* 41=175 1B5 9>45@5>45>D ?6 8(&( 3D9F1D9?> ?6 (& 9> B5C@?>C5 D? )* 9C F5BI B1@94 ?33EBB9>7 G9D89> =9>ED5C 97
(134) 1>4
(135) G85B51C D85 9>3B51C5 9> 8(& <5F5<C D1;5C C5F5B1< 8?EBC 1>4 9C 3?=@1B12<5 G9D8 D85 9>3B51C5 C55> 9> ! 35<<C 6?<<?G9>7 8(& ?F5B5H@B5C C9?> B5CE<D9>7 9> 3853;@?9>D CE@@B5CC9?> 97
(136) 1>4 CE@@<5 =5>D1< 97 ' (85C5 ;9>5D93C 1B5 3?>C9CD5>D G9D8 D85 9451 D81D 35<< 3I3<5 9>8929D9?> 81C D? 25 B1@94 G89<5 D85 B53?F5BI D1;5C C5F5B1< 8?EBC 45@5>49>7 ?> D85 5HD5>D ?6 41=175 1C54 ?> D85C5 69>49>7C ?EB B5CE<DC 9=@<I 1 =?45< G85B5 E@B57E<1D9?> ?6 8(& 9> B5C@?>C5 D? )* 3?>CD9DED5C 1 6554213; <??@ 2B9>79>7 4?G> (& 13D9F9DI D? B59>9D91D5 35<< 3I3<5 @B?7B5CC9?> 97 #EB B5CE<DC C8?G D81D B54E3D9?> 9> 5>4?75>?EC 8(& <5F5<C <514C D? (& 13D9F1D9?> G9D8?ED D85 9>4E3D9?> ?6 1@@1B5>D " 41=175 (85C5 B5CE<DC @B?=@D54 EC D? 9>F5CD971D5 G85D85B (& 1>4 8(& 9>D5B13D 49B53D<I >D5B5CD9>7<I 8?=?<?7E5C ?6 (& 9> I51CD 1>4 0 !(#-.1(1 1B5 9>F?<F54 9> D5<?=5B5 =5D12?<9C= 1>4 9> I51CD !53 @ (& 8?=?<?7 1CC?391D5C G9D8 D5<?=5B5C
(137)
(138) +5 G5B5 E>12<5 D? 45=?>CDB1D5 1 49B53D 9>D5B13D9?> ?6 (& 1>4 8(& (8EC ?EB 6EBD85B 5H@5B9=5>DC C8?E<4 5<E39 41D5 2I G8938 =5381>9C= 8(& 9>6<E5>35C (& 13D9F9DI (& G1C @B5F9?EC<I C8?G> D? 25 9>F?<F54 9> C97>1<9>7 @1D8G1IC 13D9 F1D54 2I B5@<931D9?> 1BB5CD 1>4 " 41=175 1CC1E<D 9> 35<<C 9> ' 1>4 @81C5C ?==?> 6B179<5 C9D5C 1B5 ;>?G> D? 25 CEC35@D92<5 8?DC@?DC D? 38B?=?C?=1< 2B51;175 B51BB1>75 =5>D 1>4 45<5D9?> 1>4 81F5 255> 9=@<931D54 9> D85 75>?=93 9>CD129<9DI 6B5AE5>D<I ?2C5BF54 9> 31>35B
(139) (& 9C 9>F?<F54 9> =19>D19>9>7 D85 CD129<9DI ?6 6B179<5 C9D5C 1C 9DC 9>8929D9?> <514C D? 1> 9>3B51C5 9> D85 5H@B5CC9?> ?6 6B179<5 C9D5C #EB 69>49>7C 45=?>CDB1D5 D81D 8(& 5H@B5CC9?> 9>4E35C D85 1@@51B1>35 ?6 6B179<5 C9D5C D? 1> 5HD5>D C9=9<1B D? (&;4 D8EC CE775CD9>7 D81D 1> 9>3B51C5 9> 8(& <5F5<C 31> <514 D? 75>?=93 9>CD129<9DI 'E@ @?BD9>7 D89C 3?>3<EC9?> 9C ?EB ?2C5BF1D9?> D81D 1> 9>3B51C5 9> 8(& <5F5<C G51;5>C D85 ! 1BB5CD 1 3853;@?9>D 3?>DB?<<54. FIGURE 6. Model for the hTR-mediated negative feedback loop on ATR activity in response to UV. Low levels of UV radiation activate the ATR kinase early in the UV response, which phosphorylates downstream targets, among which are p53 and Chk1, leading to a cell cycle arrest and induction of DNA repair. Independently, hTR levels are increased by a yet unknown mechanism. These increased hTR levels inhibit ATR at a later stage.. 1<C? 2I (& (85C5 B5CE<DC 1<C? CE775CD D81D 8(& 5H@B5CC9?> 31> <514 D? B54E354 6945<9DI ?6 D85 (&45@5>45>D 3853;@?9>DC 1>4 D85B56?B5 D? 9>3B51C54 75>?=93 9>CD129<9DI &535>D<I D85 7B?E@ ?6 <9J125D8 <13;2EB> 81C C8?G> D81D B54E3D9?> 9> 8(& <5F5<C 9> 31>35B 35<<C 5<939DC 1 B1@94 1>D9@B? <965B1D9F5 B5C@?>C5 ?G5F5B G85> 3?=@1B9>7 8(& ;>?3; 4?G> 9> (. 35<<C G9D8 @
(140) >E<< (. 35<<C D85I 3?> 3<E45 D81D D85 7B?GD8 9>8929D?BI B5C@?>C5 9C @
(141) 9>45@5>45>D #EB B5CE<DC ?> D85 ?D85B 81>4 9>4931D5 1 @1BD91< 45@5>45>35 ?> @
(142) 6E>3D9?> #>5 ?2F9?EC B51C?> 6?B D89C 49C3B5@1>3I 31> 25 D81D 9 $2 * 21C54 D859B 3?>3<EC9?> ?> B5CE<DC ?2D19>54 G9D8 DG? (. 35<< <9>5C G9D8 F5BI 49665B5>D 7B?GD8 B1D5C D85 @
(143) >E<< 35<< <9>5 7B?GC 1> ?B45B ?6 =17>9DE45 C<?G5B D81> D85 G9<4 DI@5 35<< <9>5 =1;9>7 1 3?=@1B9C?> 25DG55> 7B?GD8 9>89 29D9?>C F5BI 496693E<D > 3?>DB1CD G5 EC54 9> ?EB CDE4I @B9=1BI 8E=1> 1>4 (
(144) 692B?2<1CDC 1C G5<< 1C ! 2B51CD 31B39 >?=1 35<<C 1>4 49B53D<I 3?=@1B54 D85= G9D8 3?BB5C@?>49>7 ;>?3;4?G> @
(145) 35<<C EBD85B=?B5 ?EB B5CE<DC 3<51B<I 9>4931D5 D81D D85 B57E<1D9?> ?6 (& 2I 8(& 9C CE669395>D D? 16653D 35<<E<1B @1D8G1IC #EB B5CE<DC C55= D? 3?>DB1CD D85 69>49>7C D81D =(& =935 81F5 >? ?2F9?EC @85>?DI@5 9> D85 69BCD 75>5B1D9?>C
(146) (85 613D D81D 69BCD 75>5B1D9?> =(& =935 1B5 <5CC @B?>5 D? 45F5< ?@9>7 C;9> DE=?BC E@?> 385=931< 31B39>?75>5C9C 31> 25 5H@<19>54 2I =9<4 13D9F1D9?> ?6 C?=5 " 41=175 3853;@?9>DC 9>4E354 2I <?CC ?6 =(&
(147) +85D85B D89C 56653D 9C =5491D54 D8B?E78 (& B5=19>C D? 25 5<E3941D54 449D9?>1<<I D85B5 1B5 C5F5B1< 49665B5>35C 25DG55> =?EC5 1>4 8E=1> D5<?=5B5 8?=5 ?CD1C9C D81D 3?=@<931D5 5HD5>49>7 69>49>7C 6B?= =?EC5 =?45<C D? D85 8E=1> C5DD9>7 9BCD =EB9>5 35<<C 81F5 5HDB5=5<I <?>7 1>4 8I@5BF1B912<5 D5<?=5B5C 1>4 D5<?=5B1C5 13D9F9DI 9C 45D53D 12<5 9> =?CD C?=1D93 D9CCE5C '53?>4 1<D8?E78 D5<?= 5B1C5 9C 13D9F1D54 1>4 =(& 9C E@B57E<1D54 (, 4(4- 9> C5F5B1< =?EC5 DE=?B =?45<C 9D 1@@51BC >?D D? 25 B5AE9B54 6?B 7B?GD8 4EB9>7 D85 35<< 49F9C9?>C >535CC1BI 6?B DE=?B 6?B=1D9?> CE7 75CD9>7 D81D =(& 1>4?B D5<?=5B1C5 81F5 1449D9?>1< 6E>3D9?>C D? D5<?=5B5 5HD5>C9?>
(148) L
(149) > 1449D9?> D5<?=5B5 4IC6E>3. 75.
(150) Chapter 4 D9?> 9> =935 1@@51BC D? 25 C?<5<I 45@5>45>D ?> @
(151) G85B51C 9> 8E=1> 35<<C D85 @& @1D8G1I 9C 1<C? 13D9F1D54 (89B4 G89<5 &" 9>D5B65B5>35 31EC5C 1 61CD B54E3D9?> 9> 8(& <5F5<C ;>?3;?ED ?6 D5<?=5B1C5 &" 9> 75B= <9>5 35<<C =1I 1<<?G 6?B 3?=@5>C1D9?> 5F5>DC D? ?33EB
(152) (8EC ?EB B5CE<DC =1I 9>49 31D5 6?B 59D85B 1 49665B5>35 9> D5<?=5B1C5 &" 29?<?7I 25DG55> =935 1>4 =5> ?B D? 49665B5>35C 9> =5D8?4C EC54 EBD85B 5H@5B 9=5>DC G9D8 =EB9>5 35<<C 1B5 B5AE9B54 D? 5CD12<9C8 G85D85B =(& 1<C? B57E<1D5C (& 1CD ?EB B5CE<DC =1I @B?F945 1> 5H@<1>1D9?> G8I 9> D85 F1CD =1:?B9DI ?6 C?=1D93 8E=1> 35<<C 8(& 9C E29AE9D?EC<I 5H@B5CC54 G85B51C 2?D8 8(&( 1>4 D5<?=5B1C5 13D9F9DI 1B5 =?CD<I 12C5>D 8(& B57E<1D5C " 41=175 @1D8G1IC 9> 1 D5<?=5B1C5 1>4 8(&(9>45@5>45>D =1>>5B #EB B5CE<DC D8EC 31> 5H@<19> ?2C5BF1D9?>C CE775CD9>7 D81D 8(& @<1IC 1 B?<5 9> D85 9>9D91D9?> ?6 DE=?B975>939DI 1>4 D81D 9D 9C 6B5AE5>D<I E@B57E<1D54 9> 8E=1> 31>35B 35<< <9>5C
(153) L
(154)
(155) "),-5*$#&+$,219 $ 2' ,) ** +$+!$01 -% 2'$ & +( * !-0 2-07 %-0 12(+3* 2(,& #(1"311(-,1 ,# 2$4$ ")1-, ,#
(156) 21'$4 $0$+ %-0 0$ &$,21. REFERENCES *A/52,4 *7- 88;1.*- "
(157) Exp. Cell Res. 25, C
(158) 870 ( " &;201= & *7- "1*A & Microbiol. Mol. Biol. Rev. 66, C =*+5. 8/ ,87=.7=< *;5.A >=,1.; *7- ;.2-.; & Nature 345,. C
(159) . .6*77 # "=;870 *8 ( *7- ;.2-.; & Cell 107,
(160) C -D--* -2 *0*07* !.*9.; 5*A*;;*,. 2.05.; *;; %87 )05272,42 # "*;.=B42 *;=.; *7- *,4<87 " Nature 426, C
(161) "=.@*;= " .7 8;*=1 *;.A % D8778; *17 & *7- &.27+.;0 ! Nat. Genet. 33, C
(162) #*4*2 "6808;B.@<4* *7- -. *70. # Curr. Biol. 13, C
(163) .;+20 $ 8+5270 & 1.7 1.7 *7- ".-2?A Mol. Cell 14, C "1*A & .;.2;*"62=1 *7- &;201= & Exp. Cell Res. 196, C 26 & 2*=A<B.4 ;8@<. ! *;5.A &.<= 8 8?2.558 &;201= & &.27;2,1 " *7- "1*A & Science 266, C "1*A & *7- *,,1.==2 " Eur. J. Cancer 33, C ;A*7 # 705.B8> >9=* *,,1.==2 " *7- !.--.5 ! ! EMBO J. 14, C ;.2-.; & *7- 5*,4+>;7 Cell 43 C 2707.; >01.< # ! "1.?,1.748 *77 >7-+5*- % *7.,1 # ! Science 276,
(164) C
(165) *<>=862 (> ( 1>;=< " .7 8;*=1 >;;2.; .=B ;884< & *7.48 " >;*4*62 " .*9;28 &.27+.;0 ! "=.@*;= " *7- *17 & Cell 114, C
(166) &;201= & 2*=A<B.4 !*27.A & A;- & *7- "1*A &
(167) Dev. Genet. 18, C 8>7=.; ?25287 ..>?;. "=.@*;= ;.2-.; & *;5.A *7- *,,1.==2 " EMBO J. 11, C .70 >74 & &*70 " " &.27;2,1 " ?25287 12> -*6< ! ! 1*70 55<899 ! (> . " &.<= & *;5.A 7-;.@< & ;.2-.; & *7- %255.987=.*> Science 269,
(168) C 1.7 5*<,8 *7- ;.2-.; & Cell 100, C . 76. 2=,1.55 ! &88- *7- 85527< Nature 402, C %>552*6A # *;;87. 85-6*7 .*;58?. .<<5.; *<87 *7- 84*5 Nature 413, C 12*70 ( .6*77 # *=1,8,4 " #.<<*;8558 .20.7+*>6 *17 & *7- 8-.< ! Mol. Cell. Biol. 24, C "8-.; 8*;. " >2; " 8270 *;427<87 *7- .2=1 & Oncogene 14, C ;8@7 *B-*; &12=. ;- (*<126* "1*A & *7!><1270 J. Neuropathol. Exp. Neurol. 56, C !><1270 (*<126* ;8@7 &12=. "1*A & !2<<.; ! *7- *B-*; J. Neuropathol. Exp. Neurol. 56, C
(169)
(170) 8;*5.< .. *7- "1*A & Cancer 83,
(171) C
(172) *2=;* (*<126* !*=12 #26687< !80.;< "1*A & *7- *B-*; Cancer 85, C (*<126* 2=B4A *2<.; !80.;< # *6 " &2<=>+* 25,10;>+ " ";2?*<=*?* " 2*=A<B.4 "1*A & *7- *B-*; Cancer Res. 57, C (*<126* 25,10;>+ " 855*187 " *2=;* "*+88;2*7 "1*A & *7- *B-*; Clin. Cancer Res. 4, C. (2 ' #.<6.; % "*?;.#;*27 "1*A & *7- &;201= & Mol. Cell. Biol. 19, C 86. " 8,4185- 2 " *4.; " ;..7 .;56*7 255 *7- ;.<58@ J. Clin. Oncol. 23, C. 5*<,8 !2B.7 ;.2-.; & *7- *7*1*7
(173) Nat. Genet. 12, C. ;8,,852 8-5.A 87.18@.; %*;6>< *7- -. *70. #
(174) Mol. Cell. Biol. 16,
(175) C . 5*<,8 .. & *7-. "*69.; *7<-8;9 . 2718 ! *7- ;.2-.; & Cell 91, C. 87B*5.B">*;.B "*69.; 58;.< *7- 5*<,8 Nat. Genet. 26, C
(176) "*0. 255.; .;.B *7,.;* &A<8,42 *7- *,4< # Nature 424, C -D--* -2 *0*07* #.8 " *7- *,4<87 " Genes Dev. 18, C #*4*=* *781 ( >70. "12;*120. *7- *=<>>;* Mol. Cell 14, C %.;->7 ! ;*++. *00+586 *7- *;5<.-.; Mol. Cell 20, C
(177) . "*7,*; 27-<.A85=B $7<*5*,6*B *7- 277 " Annu. Rev. Biochem. 73, C . *;5<.-.; 84. 2;B8.?* *44.72<= *<=*7 .= ;272 *7- -. *70. # PLoS Biol. 2, . ;8@7 *7- *5=268;. Genes Dev. 17,
(178) C
(179) . %.<9* 8>?255287 "9*705.; *7- "1299.7 Genes Dev. 19, C. *17 & "=.@*;= " ;884< & (8;4 " *=87 >;*,12 .23.;<+.;0.7 ! 7855 .A.;<87 *7- &.27+.;0 ! Nat. Med. 5,
(180) C . .;+.;= 2==< *4.; " *625=87 " &;201= & "1*A & *7- 8;.A ! Proc. Natl. Acad. Sci. U. S. A. 96,
(181) C .
(182) 2 " ;8=1.;< *:: *7- 5*,4+>;7 J. Biol. Chem. 280, C . 2 " !8<.7+.;0 873*,8>; 8=,1427* 86 ( >71* ! *7- 5*,4+>;7 Cancer Res. 64, C . ;>66.54*69 # ! .;7*;-< ! *7- 0*62 ! Cancer Cell 2, C. %88;18.?. *7- 0*62 ! Cancer Cell 4, C !20*>= "1.?,1.748 !>=B &256 *77 *7- ".;*9127 Nat. Biotechnol. 17, C 0*62 ! *7- .;7*;-< ! Cell 102, C
(183)
(184) >>;<6* *7- 0*62 ! Mol. Cell. Biol. 25,
(185) C
(186) . 26 & *7- &> Nucleic Acids Res. 25, C . ",1.;; *==6.; ",1>5=1.2< *7<.; *7- -.; Gene Ther. 12, C ;A*7 # 705.B8> *55* 8BB* >71*6 *7- !.--.5.
(187) ATR regulation by hTR ! ! Nat. Med. 3, C.
(188) ",1..5 ",1*./.; *>,1 .55.; &*2 ;274<,162-= ?*7 %*5.7 8.,4.; & 8,418;7@8;72,B*4 *7- 8;.6+* Oncogene 20, C . ;>66.54*69 # ! .;7*;-< ! *7- 0*62 ! Science 296, C ><+A .2<=;2=B +;*1*6 ! # *;72=B *7- "*;4*;2* Cancer Res. 60, C ;*?.< ! (> ",1@*;B *5.< "*><?255. D8778; *7- 2@72,*&8;6< J. Biol. Chem. 275,
(189) C
(190)
(191) #2++.==< ! " ;>6+*>01 &2552*6< "*;4*;2* 52+A & "12.1 " ( #*A* ( ;2?.< *7- +;*1*6 ! # Genes Dev. 13, C
(192) )1*8 *7- 2@72,*&8;6< Mol. Cell. Biol. 21, C
(193) .//.;7*7 # "269<87 ;*74 ! .2758=1 *>5.< ! " 8;-.2;8"=87. *7- *>/6*77 & Mol. Cell. Biol. 22,. C
(194)
(195) ",1@*;=B )58=8;A7<42 *7- .;.6
(196) Cancer Lett. 232, C
(197)
(198) *<9.; 012.6 ;5= *7- 58?.; # & Cell 111, C
(199) !2=,12. *558;A *7- .=.< # Mol. Cell. Biol. 19,
(200)
(201) C
(202)
(203)
(204) #*4*=* #*7*4* ( *7- *=<>>;* Mol. Cell 17, C
(205) "1.,1=.; 8<=*7B8 % *7- *>=2.; Nat. Cell Biol. 6,
(206) C
(207)
(208) 295270 *7- 884. Nature 347, C
(209) ;8@<. ! *7- ;.2-.; & Proc. Natl. Acad. Sci. U. S. A. 92,. C "6808;B.@<4* *7- -. *70. # EMBO J. 21, C. 127 ;=*7-2 " "1.7 #*6 .. " 8==52.+ ;.2-.; & *7- . 2718 ! Cell 97, C . 77.
GERELATEERDE DOCUMENTEN
In chapter 4 I have investigated the role of hTR, the RNA component of telomerase (an enzyme involved in the acquired capability of cancer cells to reach a limitless
2 Dnd1 function is essential for proper development of primordial germ cells (PGCs) in zebrafish and mammals, indicating a crucial role for RBP/miRNA interplay on 3’UTRs of mRNAs
Here we demonstrate that the expression of dead end 1 Dnd1, an evolutionary conserved RNAbinding protein RBP, counteracts the function of several miRNAs in human cells and in
To assess whether increasing the miR-148 level would result in a reduced endogenous DNMT3b1 protein level, we electroporated the miR-148 expression vector into 2102EP cells
In chapter 3, I have described the regulatory role of the RNA binding protein Dead end 1 in the development of germ cells, as we found in zebrafish model systems and human
Tot nu toe hebben alle geïdentificeerde humane boodschapper-RNA’s die door microRNA’s worden gereguleerd hun bindingsplaatsen voor deze moleculen aan een van de uiteinden van
Zijn fascinatie voor onderzoek naar kanker kreeg een eerste impuls tijdens zijn eerste stage, op de afdeling Experimentele Hepatologie in het Academisch Medisch Centrum in
Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/13414 Note: To
