US 200400185.50A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0018550 A1 Bellacosa (43) Pub. Date: Jan. 29, 2004 (54) ANTIBODIES IMMUNOLOGICALLY continuation-in-part of application No. 09/463,891, SPECIFIC FOR A DNA REPAIR filed on Jan. 28, 2000, now abandoned, filed as 371 of ENDONUCLEASE AND METHODS OF USE international application No. PCT/US98/15828, filed THEREOF on Jul. 28, 1998. (76) Inventor: Alfonso Bellacosa, Philadelpha, PA Publication Classification (US) (51) Int. Cl." ............................. C12Q 1/68; CO7H 21/04 Correspondence Address: DANN, DORFMAN, HERRELL & SKILLMAN (52) U.S. Cl. ............................................... 435/6; 536/24.3 1601 MARKET STREET SUTE 2400 PHILADELPHIA, PA 19103-2307 (US) (57) ABSTRACT (21) Appl. No.: 10/629,951 An isolated nucleic acid molecule encoding a human DNA repair enzyme, MED1, is disclosed. Like other mismatch (22) Filed: Jul. 29, 2003 repair genes which are mutated in certain cancers, MED1, encoding nucleic acids, proteins and antibodies thereto may Related U.S. Application Data be used to advantage in genetic or cancer Screening assayS. MED1, which recognizes and cleaves DNA, may also be (63) Continuation of application No. 09/629,222, filed on used for the diagnostic detection of mutations and genetic Jul. 31, 2000, now Pat. No. 6,599,700, which is a variants Patent Application Publication Jan. 29, 2004 Sheet 1 of 44 US 2004/0018550 A1 LexA-MLH1 / B42-f5 LeXA/B42-f5 LexA-myc / B42-f5 LeXA-bicoid / B42-f5 LeXA-K-rev 1 / B42-f5 LeXA-K-reV-1 / Krit Leu- X-gal Fig. I Patent Application Publication Jan. 29, 2004 Sheet 2 of 44 US 2004/00185.50A1 3 St C - O 1.35 -> Fig. 2 Patent Application Publication Jan. 29, 2004 Sheet 4 of 44 US 2004/0018550 A1 S. A e. > a > a as a C EY pa M. A () E r. se on U A KD (9 - - r a () (r. A (5 n, as a > - 2 <d r ( E- a la s SC I ap r - a () ?h > o d- 2. r (CH s () a a O. a CD (na > - A > A 4 A d Y - it H - (5) r I a. r () > a r C a O. a. O. N. Sea 3. A "O5 O.i "O ;C, "O O, Patent Application Publication Jan. 29, 2004 Sheet 5 of 44 US 2004/0018550 A1 a - E st U. - to a is a bar Z. a -- a - - 4 H as A as as a ir - - 2 O ( 2. appa as a as CD yer E 55wr vs.r- g er re EE5 sh w e 5'EE:o EEE5 ran to y > rhBE to g r E:5to S. C. As Oto b E) o U to O C. A Patent Application Publication Jan. 29, 2004 Sheet 6 of 44 US 2004/0018550 A1 3 E.s c E as Eaa Uni a - D > s > H Sc Cl D s E r str ), a a CD U - La - - Ca a CD C) U 2. K. Y i. He I - - r a A Patent Application Publication Jan. 29, 2004 Sheet 7 of 44 US 2004/0018550 A1 Fig. 5 Patent Application Publication Jan. 29, 2004 Sheet 8 of 44 US 2004/0018550 A1 s L > ch CO CC CC 2 Z ?h ?h O CD O Ol KD 144 87 32.7- Fig. 6 Patent Application Publication Jan. 29, 2004 Sheet 9 of 44 US 2004/0018550 A1 HCOO Patent Application Publication Jan. 29, 2004 Sheet 10 of 44 US 2004/0018550 A1 Fig. 7B Patent Application Publication Jan. 29, 2004 Sheet 11 of 44 US 2004/0018550 A1 Fig. 8 Patent Application Publication Jan. 29, 2004 Sheet 12 of 44 US 2004/0018550 A1 i s s O C) N - ( C) Patent Application Publication Jan. 29, 2004 Sheet 13 of 44 US 2004/0018550 A1 Fig. 10A Patent Application Publication Jan. 29, 2004 Sheet 14 of 44 US 2004/0018550 A1 MBD: - -- - - - - - - -- Cold methylated: - - - - -- - Cold unmethylated: - - + - - -- Bound-o- 8P-methylated 3P-unmethylated Fig. 10B Patent Application Publication Jan. 29, 2004 Sheet 15 of 44 US 2004/00185.50A1 -- hvSH2 total lysate IP: anti-FLAG Western: anti-hvSH2 Fig. 11A Patent Application Publication Jan. 29, 2004 Sheet 16 of 44 US 2004/0018550 A1 total lysate IP: anti-FLAG Western: anti-FAG Fig. 11B Patent Application Publication Jan. 29, 2004 Sheet 17 of 44 US 2004/0018550 A1 Fig. 1 FC total lysate P: anti-HT HT-MED1/CMV5: CMV5: MLH1-- HT-MED1-- Patent Application Publication Jan. 29, 2004 Sheet 18 of 44 US 2004/0018550 A1 SIZI‘313N 9}}|.9||H8EB|8||01JH| Patent Application Publication Jan. 29, 2004 Sheet 19 of 44 US 2004/0018550 A1 O Y4 3 Ois H do r O O O O CO O N OO r CO s & d .9 S O CD O) CD t S C Fig. 13 Patent Application Publication Jan. 29, 2004 Sheet 20 of 44 US 2004/0018550 A1 c) CD D Y d 9C E O s 23.1 kbp - 9.4 - Fig. 14A Fig. 14B Patent Application Publication Jan. 29, 2004 Sheet 21 of 44 US 2004/0018550 A1 s & Fig. 15. Patent Application Publication Jan. 29, 2004 Sheet 22 of 44 US 2004/0018550 A1 CAAGGAAGAT ATTGCIGTTG GACTGGGAGG AGTGGGAGAA GATGGAAAGG 51 ACCTGGTGAT AAGCAGTGAG CGCAGCTCCC TTCTCCAAGA GCCCACTGCT 101 TCTACTCTGT CTACTACTAC AGCGACAGAA GGCCACAAGC CTGTCCCGTG 151 TGGATGGGAA AGAGTTGTGA AGCAAAGGIT ATCTGGGAAA ACTGCAGGAA 201 AATTTGATGT ATACTITATC AGCCCACAAG GATTGAAGTT CAGATCAAAA 251 CGTTCACTTG CTAATTATCT TCTCAAAAAT GGGGAGACTT TTCTTAAGCC 301 TGAAGATTTT AATTTTACTG TACTGCCGAA AGGGAGCATC AATCCCGGTT 351 ATAAACACCA AAGTTTGGCA GCTCTGACTT CCCTGCAGCC AAATGAAACT 401 GACGTTTCAA AGCAGAACCT CAAGACACGA AGCAAGTGGA AAACAGATGT 451 GTTGCCTCTG CCCAGTGGTA CTTCAGAGTC GCCAGAAAGC AGCGGACTGT 501 CTAACTCTAA CTCGGCTTGC TTGCTATTGA GAGA ACATAG GGACATTCAG 591 GATGTTGACT CTGAGA AGAG GAGAAAGTCC AAAAGAAAGG TGACTGTTTT 601 GAAAGGAACT GCAAGTCAGA AAACCAAACA AAAGTGCAGG AAGAGTCTCT 651 TAGAGTCTAC TCAAAGAAAC AGAAAAAGAG CATCTGTGGT TCAGAAGGTG 701. GGTGCTGATC GCGAGCTGGT GCCACAGGAA AGTCAACTCA ACAGAACCCT 751. CTGCCCTGCA GATGCCTGTG CAAGGGAGAC TGTTGGCCTG GCTGGGGAAG 801 AAAAATCACC AAGCCCAGGA CTGGATCTTT GTTTCATACA AGTAACTTCT 851 GGCACCACAA ACAAATTCCA TTCA ACTGAA GCAGCAGGTG AAGCAAATCG 901 TGAGCAGACT TTTTTAGAAT CAGAGGAAAT CAGATCGAAG GGAGACAGAA 951 AGGGGGAGGC ACATTTGCAT ACTGGTGTTT TACAGGATGG CTCTGAAATG 1001 CCCAGCTGCT CACAAGCCAA GAAACACTTT ACTTCTGAGA CATTTCAAGA 1051 AGACAGCATC CCACGGACAC AAGTAGAAAA AAGGAAAACA AGCCTGTATT 1.101 TTTCCAGCAA GTACAACAAA GAAGCTCTTA GCCCCCCAAG ACGCAAATCC 1151 TTCAAGAAAT GGACCCCTCC TCGGTCACC TTTAATCTTG TTCAAGAAAT 120 ACTTTTCCAT GACCCATGGA AGCTCCTCAT CGCGACTATA TTTCTCAATC 1251 GGACCTCAGG CAAGATGGCC ATCCCTGTGC TGTGGGAGTT TCTAGAGAAG 1301 TACCCTTCAG CTGAAGTGGC CCGAGCTGCC GACTGGAGGG ACGTGTCGGA Fig. 6A Patent Application Publication Jan. 29, 2004 Sheet 23 of 44 US 2004/0018550 A1 1351 GCTTCTCAAG CCTCTTGGTC TCTACGATCT CCGTGCAAAA ACCATTATCA 1401, AGTTCTCAGA TGAATATCTG ACAAAGCAGT GGAGGTATCC GATTGAGCTT 1451 CATGGGATTT GGTTAAAATA TGGCAACGAC TCTACCGGAT. CTTTTGTGTC 1501 AATGAATGGA ACAG Fig. 16B Patent Application Publication Jan. 29, 2004 Sheet 24 of 44 US 2004/0018550 A1 mouse MED1 protein (upper sequence) X human MED1 protein (lower sequence) 1. KEDIAVGLGGVGEDGKDLVI. SSERSSLLQEPTAST. LSSTTATEGHKP A 7 36 KEDVAMELERVGEDEEOMMIKRSSECNPLLOEPIASAOFGATAGTECRKS 85 48 VPCGWERVVKQRLSGKTAGKFDVYFISPOGLKFRSKRSLANYLLKNGETF 97 | | | | | | | | | | | | | | | | | | : | | | | | | | | | | | | | | | | | | | | | | | | | | 86 VPCGWERVVKORLFGKTAGRFDVYFISPQGLKFRSKSSLANYLHKNGETS 135 98 LKPEDFNFTVLPKGSINPGYKHQSLAALTSLOPNETDVSKQNLKTRSKWK 147 | | | | | . || | | | | | : : . : 136 LKPEDFDFTVLSKRGIKSRYKDCSMAALTSHLONQSNNSNWNLRTRSKCK 185 148 TDVLPLPSGTSESPESSGLSNSNSACLLLREHRDIQDVDSEKRRKSKRKV 197 | | | | | | | | | | | | | | | | : : ||. | | | || 186 KDVFMPPSSSSELOESRGLSNFTSTHLLLKEDEGVDDVNFRKVRKPKGKV 235 198 TVLKGTASOKTKOKCRKSLLESTORNRKRAS. - - - - - - - - - - - - - 228 236 TILKGIPIKKTKKGCRKSCSGFVQSDSKRESVCNKADAESEPVAQKSQLD 285 229 . EDSIPRTOVEKRKTSLYFSSKYNKEALSPPRRKSF 263 . ; ; | | | | | | | | | | | | | | | | | . 386 CSPTRKDFTGEKIFQEDTIPRTOIERRKTSLYFSSKYNKEALSPPRRKAF 435 264 KKWTPPRSPFNLVOEILFHDPWKLLIATIFLNRTSGKMAIPVLWEFLELY 313 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 436 KKWTPPRSPFNLVOETLFHDPWKLLIATIFLNRTSGKMAIPVLWKFLEKY 485 314 PSAEVARAADWRDVSELLKPLGLYDLRAKTIIKFSDEYLTKQWRYPIELH 363 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | : | | | | | | | | | |: 486 PSAEVARTADWRDVSELLKPLGLYDLRAKTIVKFSDEYLTKQWKYPIELH 535 364 GIWLKYGNDSYRIFCVNEWKO 384 | | | | | | | | | | | | | | | 536 GIG. KYGNDSYRIFCVNEWKQ 555 Fig. 17 Patent Application Publication Jan. 29, 2004 Sheet 25 of 44 US 2004/0018550 A1 Fig. 18A Exon 2 ggttttgtttittccago'AAGGAAGATATTGCTGTTGGACTGGGAGGAGTG GGAGAAGATGGAAAGGACCTGGTGATAAGCAGTGAGCGCAGCTCCCTTCT CCAAGAGCCCACTGCTTCTACTCTGTCTAGTACTACAGCGACAGAAGGCC ACAAGCCTGTCCCGTGTGGATGGGAAAGAGTTGTGAAGCAAAGGTTATCT GGGAAAACTGCAGGAAAATTTGATGTATACTTTATCAGgtaagcatttag Galaggaaaata Fig. 18B Exon 3 cttttttittttitccttittaagCCCACAAGGATTGAAGTTCAGATCAAAAC GTTCACTTGCTAATTATCTTCTCAAAAATGGGGAGACTTTTCTTAAGCCT GAAGATTTTAATTTTACTGTACTGCCGAAAGGGAGCATCAATCCCGGTTA TAAACACCAAAGTTTGGCAGCTCTGACTTCCCTGCAGCCAAATGAAACTG ACGTTTCAAAGCAGAACCTCAAGACACGAAGCAAGTGGAAAACAGATGTG TTGCCTCTGCCCAGTGGTACTTCAGAGICGCCAGAAAGCAGCGGACTGTC TAACTCTAACTCGGCTTGCTTGCTATTGAGAGAACATAGGGACATTCAGG ATGTTGACTCTGAGAAGAGGAGAAAGTCCAAAAGAAAGGTGACTGTTTTG AAAGGAACTGCAAGTCAGAAAACCAAACAAAAGTGCAGGAAGAGTCTCTT AGAGTCTACTCAAAGAAACAGAAAAAGAGCATCTGTGGTTCAGAAGGTGG GTGCTGATCGCGAGCTGGTGCCACAGGAAAGTCAACTCAACAGAACCCTC TGCCCTGCAGATGCCTGTGCAAGGGAGACTGTTGGCCTGGCTGGGGAAGA AAAATCACCAAGCCCAGGACTGGATCTTTGTTTCATACAAGTAACTTCG GCACCACAAACAAATTCCATTCAACTGAAGCAGCAGGTGAAGCAAATCGT GAGCAGACTTTTTTAGAATCAGAGGAAATCAGATCGAAGGGAGACAGAAA GGGGGAGGCACATTTGCATACTGGTGTTTTACAGGATGGCTCTGAAATGC CCAGCTGCTCACAAGCCAAGAAACACTTTACTTCTGAGACATTTCAAGgt actCagtgcatgaaaa Patent Application Publication Jan. 29, 2004 Sheet 26 of 44 US 2004/0018550 A1 Fig. 18C EKon 4 gactataaactaattittgct tctoagAAGACAGCATCCCACGGACACAAG TAGAAAAAAGGAAAACAAGCCTGTATTTTTCCAGCAAGTACAACAAAGAA Ggtaccoacct ttccctaagc Fig. 18D Eko. 5 tatatttntgnagCTCTTAGCCCCCCAAGACGCAAATCCTTCAAGAAATG GACCCCCCTCGGTCACCTTTAATCTTGTTCAAGAAATACTTTTCCATG ACCCATGGAAGCTCCTCATCGCGACTATATTTCTCAATCGGACCTCAGg ttingggg.tcattgncat Fig. 18E Exon 6 tgtt tatgctccc.cagoCAAGATGGCCATCCCTGTGCTGTGGGAGTTTCT AGAGAAGTACCCTTCAGCTGAAGTGGCCCGAGCTGCCGACTGGAGGGACG