Supplemental Materials and Methods:

Transcription

1 Supplemental Materials and Methods: Cloning: Oligonucleotides used in the subcloning steps are listed in Supplemental Table 1. Human FANCI (isoform 1, KIAA1794) was subcloned from pcmv6-xl4 [FANCI] in two steps: PCR primers with a 5 BamHI linker (FIN-BamHI F) and a 3 SalI linker (FIN-SalI R) were used to amplify FANCI N-terminal coding sequence (codons 1-439) and ligated with BamHI, SalI-digested pfastbacht(b) (Invitrogen). C-terminal FANCI coding sequence was added to this construct as a NotI-NsiI fragment from pcmv-xl4[fanci] to generate full-length FANCI with an N-terminal 6xHis epitope. The FANCI K523R mutation was generated by Quickchange mutagenesis using primers FANCI K523R F and FANCI K523R R. Human FANCL was subcloned from pmieg3his/flag-fancl by PCR with primers FANCL TopoF and FANCL TopoR. The PCR product was inserted into pentr/d-tev-topo, and then transferred into pdest20 by site-specific recombination with LR Clonase (Invitrogen) to generate FANCL with an N-terminal GST epitope. The FANCL C307A mutation was generated by Quickchange mutagenesis using primers FANCL C307A F and FANCL C307A R. Bacmids harboring the expression constructs above were derived from all plasmids in DH10Bac cells according to the manufacturer s instructions (Invitrogen). Sf9 insect cells (Invitrogen) were transfected with bacmids to produce baculoviruses, as well as to amplify the baculoviral titer. Human UBE2T was subcloned from potb7-hube2t (ATCC, Manassas, VA). The PCR product was restricted with BamHI and NotI and ligated with BamHI-, NotIrestricted pgex-6p-1 (GE Healthcare) to generate UBE2T with an N-terminal GST epitope. pgex-6p-1-hube2t was used as a template for Quickchange mutagenesis with primers UBE2T C86A F and UBE2T C86A R to generate the UBE2T C86A mutation. Two C-terminal fragments of FANCI (FIΔN1: codons , and FIΔN2: codons ), were generated by PCR using oligonucleotides FANCI 985 F and FANCI 965 F, respectively, with FANCI NotI R. The BamHI-NotI-restricted PCR product was ligated with BamHI-NotI-restricted pgex-6p-1 to generate FIΔN1 and FIΔN2, each with an N-terminal GST epitope. 1

2 Protein expression and purification FANCI, FANCI K523R and FANCL were produced in High-Five insect cells (Invitrogen) by infection, or co-infection, with the appropriate baculovirus(es) for hours at 27 C, whereupon cells were harvested by centrifugation. UBE2T, FIΔN1 and FIΔN2 were produced in E. coli (Rosetta DE3 plyss), inducing with 0.1 mm IPTG for 6 hours at 16 C for UBE2T, and for hours for the GST-FANCI fragments. Cell pellets were stored at 80 C. Purification of FANCI and FANCI K523R All the purification steps were carried out at 4 o C. Insect cells (300 ml culture) infected with the FANCI or FANCI K523R baculovirus were lysed in 50 ml CBB buffer (50 mm Tris-HCl, ph 7.5, 10% sucrose, 2 mm EDTA) supplemented with 200 mm KCl, 0.01% Igepal (Sigma), 1mM β-mercaptoethanol, and protease inhibitors (5 µg/ml each of leupeptin, chymotrypsinogen, aprotinin, and pepstatin, 0.1 mm PMSF, and 1mM benzamidine), and sonicated for 30 seconds twice on a Bransen 250 sonifier set to power 4.5, 50% output. The lysate was subjected to ultracentrifugation for 90 min at 100,000 Xg. The supernatant was diluted with two volumes of T buffer (25 mm Tris-HCl, 10% glycerol, 0.5 mm EDTA, 0.01% Igepal, 1mM β-mercaptoethanol, and protease inhibitors named above), and then loaded onto a Q Sepharose Fast Flow (Amersham Biosciences) column (40 ml) equilibrated with T buffer containing 100 mm KCl. The column was washed with 100 ml of T buffer containing 100 mm KCl, then eluted with a 400-ml gradient from 150 mm to 550 mm KCl in T buffer. Fractions containing His-FANCI of FANCI K523R eluting between ~ mm KCl were pooled and mixed with 2 ml of Nickel-NTA beads (Qiagen) for 2 hours. The beads were washed with 25 ml each of T buffer containing 1M KCl and 15 mm imidazole, and T buffer containing 100 mm KCl and 30 mm imidazole, before being treated with ml of T buffer with 100 mm KCl containing 300 mm imidazole to elute the bound proteins. The eluate fractions were pooled and fractionated with a 1 ml Mono Q column with a 30 ml, mm KCl gradient in T buffer. Fractions containing (His) 6 -FANCI, or (His) 6 -FANCI K523R, eluting between ~ mm KCl were pooled and concentrated to ~10 mg/ml using an 2

3 Ultracel-30K concentrator (Amicon) before being frozen in liquid nitrogen in small aliquots and stored at 80 C. Purification of FIΔN1 and FIΔN2 All the steps were performed at 4 C. GST-FIΔN1 and GST-FIΔN2 were purified from ~20 g of E. coli paste lysed in 100 ml of T buffer containing 250 mm KCl, 0.01 % Igepal, 1 mm DTT and protease inhibitors as above. Lysates were sonicated for 4 minutes with a Bransen 250 sonifier set to power 7, 50% output, and then subjected to ultracentrifugation for 90 min at 100,000 Xg. The supernatant was diluted with two volumes of T buffer and then loaded onto a Q Sepharose Fast Flow (Amersham Biosciences) column (35 ml) equilibrated with T buffer containing 100 mm KCl. The column was washed with 100 ml of T buffer with 100 mm KCl, then eluted with a 350- ml gradient from 100 mm to 550 mm KCl in T buffer. Fractions containing GST-FIΔN1 or GST-FIΔN2, eluting between ~ mm KCl, were pooled, supplemented with Igepal to 0.05%, and mixed with 1 ml of Glutathione-Sepharose beads (Amersham Biosciences) overnight. The beads were washed with 40 ml of T buffer containing 1 M KCl, and 5 ml of T buffer containing 100 mm KCl, before being treated with 5 ml of T+100 containing 25 mm reduced glutathione to elute the bound proteins. The eluate fractions were pooled and fractionated in a 1 ml Mono Q column with a 20-ml, mm KCl gradient in T buffer; fractions containing GST-FIΔN1, eluting between ~ mm KCl, or GST-FIΔN2, eluting between mm KCl, were pooled and concentrated to ~2 mg/ml using an Ultracel-30K concentrator before being frozen in liquid nitrogen in small aliquots and stored at 80 C. Purification of FANCL and FANCL C307A All the purification steps were carried out at 4 C. Insect cells (380 ml culture) infected with the GST-FANCL or GST-FANCL C307A baculovirus were lysed in 50 ml CBB buffer containing 300 mm KCl, sonicated and clarified as above. The clarified lysate was diluted with T buffer to 60mM KCl, then loaded onto a 40 ml Q Sepharose Fast Flow (Amersham Biosciences) column equilibrated with T buffer containing 100 mm KCl. The column was washed with 100 ml of T buffer containing 100 mm KCl, and then 3

4 eluted with a 400 ml gradient of mm KCl. Fractions containing GST-FANCL or GST-FANCL C307A eluting between 250 and 430 mm KCl were pooled, supplemented with Igepal to 0.1% and KCl to 500 mm, and then mixed with 4 ml Glutathione-Sepharose beads for 2 hours at 4 C. The affinity beads were washed with 30 ml each of T buffer with 500 mm KCl and T buffer with 1 M KCl before being treated with 24 ml T buffer with 100 mm KCl and 25 mm glutathione. The eluate was applied onto a 1 ml Mono Q column equilibrated with T buffer containing 100 mm KCl and eluted with a 40 ml gradient of 150 to 550 mm KCl. Fractions containing GST-FANCL or GST-FANCL C307A eluting between 250 and 440 mm KCl were pooled and concentrated in a Ultracel-30K concentrator to ~1 mg/ml, frozen in liquid nitrogen in small aliquots, and stored at 80 C. Purification of UBE2T and UBE2T C86A All the purification steps were carried out at 4 C. E. coli cells expressing UBE2T or UBE2T C86A (25 g paste) were lysed in 125 ml CBB buffer containing 500 mm KCl with 0.1% Igepal, sonicated and clarified as for FIΔN1 and FIΔN2 above. The cleared lysate was diluted with an equal volume of T buffer containing 500 mm KCl and then mixed with Glutathione-Sepharose beads for 2 hours. The affinity beads were washed with 50 ml each of T buffer with 500 mm KCl and T buffer with 1 M KCl, before being treated with 18 ml T buffer with 100 mm KCl and 25 mm reduced glutathione. The eluate was passed through a 6 ml source Q column equilibrated with T buffer with 100 mm KCl. GST-UBE2T or GST-UBE2T C86A from the Source Q flow through fraction was treated with Precission protease for 12 hours to remove the GST epitope. The protein mixture was filter-dialyzed in an Ultracel-5K concentrator into T buffer with 150 mm KCl, and the cleaved GST epitope and the GST-tagged Precission protease were removed by mixing with 4 ml of Glutathione Sepharose for 12 hours. The supernatant containing UBE2T or UBE2T C86A was concentrated to ~ 1 mg/ml using an Ultracel-5K concentrator, frozen in liquid nitrogen in small aliquots and stored at 80 C. 4

5 Supplemental Figure legends: Supplemental Figure 1. FANCI binds plasmid length ssdna and dsdna. Increasing amounts of FANCI ( µm) were mixed with 150 ng of viral + strand DNA (ss), 100 ng of supercoiled, double-stranded RFI (sc), and/or 100 ng of StuIlinearized double-stranded DNA (ds). Note that treatment of nucleoprotein complexes with SDS and proteinase K (SDS+PK) released the DNA substrates. Supplemental Figure 2. Binding of duplex and partial duplexes by FANCI. FANCI ( µm) was incubated with a mixture of radiolabeled double-stranded linear (H3/H4) and either 5 overhang or 3 overhang DNA (0.3 pmol each). The reaction mixtures were analyzed as in Figure 1. Supplemental Figure 3. FANCI is ubiquitinated by FANCL and UBE2T All reactions contained UBE1, in addition to the components indicated. The C86A mutation in UBE2T disrupts its E2 Ub conjugating activity. Addition of wild type fulllength human FANCI (WT) to the ubiquitination reaction containing FANCL, UBE2T and ATP results in FANCL auto-ubiquitination (Ub-FANCL) as well as a higher molecular weight product - an apparent doublet - visualized with anti-ha (Ub-FANCI). The FANCI K523R substrate (KR) produces only the lower band, with a weaker signal than the wild type protein. 5

6 SUPPLEMENTAL TABLE 1 Oligonucleotides used for subcloning Oligonucleotide name DNA sequence (5 to 3 ) FIN-BamHI F FIN-SalI R FI 985F FI 965F FANCI NotI R FANCI K523R F FANCI K523R R FANCL TopoF FANCL TopoR FANCL C307A F FANCL C307A R UBE2T BamHI F UBE2T NotI R UBE2T C86A F UBE2T C86A R CATGGGATCC GACCAGAAGA TTTTATCTCT AGCAGCAG CTCTGGTCGA CTTTCTTGTC TGATCATCTC ATGGATC CACCGGATCC CTAGTCACGG TTCTTACCAG TTTGTCC CACCGGATCC TCCTTGAATT TACTTAGCAG TCAAGAG GAAAGCGGCCGCAATCTAGAGTCGAG CCTTCGGAGA GCTATGTTTG CCAACC ACATAGCTCT CCGAAGGACA AGTATCAAG CACCATGGCG GTGACGGAAG CGAGC TCAGTGTTTC CTTCCAGACA TTTTTAAG ACTATGGATG CTGGAATTTG TTATGCTTATC CAAATTCCAG CATCCATAGT AAAATCAGAT T CACCGGATCC ATGCAGAGAG CTTCACGTCT GAAG CGAGCGGCCG CAGGACAAGT CCCCTAAACA TCAGGATG GGAAGGATTG CTCTGGATGT TCTCAAATTG CC GAACATCCAG AGCAATCCTT CCAGCAG 6

7 SUPPLEMENTAL TABLE 2A Oligonucleotides used for DNA binding substrates Oligonucleotide name DNA sequence (5 to 3 ) H1 H2 H3 H4 H5 H6 H7 H8 ATTAAGCTCT AAGCCATGAA TTCAAATGAC CTCTTATCAA CATATTTAAA ACATGTTGGA TCCCAGCACC AGATTCAGCA TTGATAAGAG GTCATTTGAA TTCATGGCTT AGAGCTTAAT TGCTGAATCT GGTGCTGGGA TCCAACATGT TTTAAATATG CATATTTAAA ACATGTTGGA TCCCAGCACC AGATTCAGCA ATTAAGCTCT AAGCCATGAA TTCAAATGAC CTCTTATCAA CATATTTAAA ACATGTTGGA TCCCAGCACC AGATTCAGCA TACGTTACCG ATCGTACGTT CGATGCTGGC TACTGCTAGC GCTAGCAGTA GCCAGCATCG AACGTACGAT CGGTAACGTA GCTAGCAGTA GCCAGCATCG AACGTACGAT CGGTAACGTA GTCGATTATC GAGATCAAGC TAGCATAGCC ATAGCGCGAC GTCGCGCTAT GGCTATGCTA GCTTGATCTC GATAATCGAC ATTAAGCTCT AAGCCATGAA TTCAAATGAC CTCTTATCAA dt (T) 83 P1 TTATATCCTT TACTTTGAAT TCTATGTTTA ACCTTTTACT TATTTTGTAT TAGCCGGATC CTTATTTCAA TTATGTTCAT 7

8 SUPPLEMENTAL TABLE 2B DNA binding substrates Substrate name Oligonucleotides used Structure Oligo-dT (dt) dt Single-strand P1 P1 Single-strand H3 H3 Single-strand H3/H4 H3, H4 Double-strand 3 OH H3, H1 3 overhang 5 OH H3, H2 5 overhang Y-fork H3, H5 fork Holliday junction (HJ) H3, H5, H7, H8 HJ 8

9 9 Longerich_Supp. Fig. 1, 2

10 10 Longerich_Supp. Fig. 3