SUPPLEMENTAL MATERIAL. Supplemental material contains Supplemental Figure Legends and Supplemental Figures 1 to

Transcription

1 SUPPLEMENTAL MATERIAL Supplemental material contains Supplemental Figure Legends and Supplemental Figures 1 to 6.

2 SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. Overview of the mechanisms by which replication blocking lesions on the leading strand can be processed. DNA lesions that block replication can be repaired by replication associated repair (A), or they can be bypassed without their actual removal by the DNA damage tolerance pathway (B). (A) Processing of replication blocking lesions by replication associated DNA repair. Stalling of the replication fork at the DNA lesion exposes single-stranded DNA after separation of DNA strands by replicative helicase. At this point, DNA lesions cannot be repaired by excision repair machinery, because the complementary DNA strand is not available. Replication fork regression leads to annealing of the template DNA strands, and repositions the DNA lesion back into the reformed parental duplex. The DNA lesion in the double stranded region is now available for processing by nucleotide or base excision repair. Following repair of the lesion and reversal of the regressed fork, replication continues on undamaged template. (B) Bypass of replication blocking lesions by mechanisms of DNA damage tolerance. DNA damage tolerance facilitates bypass of replication blocking lesion without its removal. Two major pathways of DNA damage tolerance can be distinguished: translesion synthesis and template switching. Translesion synthesis involves temporary exchange of the replicative polymerase with damage tolerant translesion DNA polymerase, which has the ability to accommodate the damaged DNA template and replicate past DNA damage. Template switching utilizes the newly sythesized DNA strand as a template for DNA synthesis. It involves structural rearrangement of the replication fork, which can be achieved by the regression of replication forks and the formation of the chicken-foot structures, or by recombination-like process. Supplemental Figure 2. (A) Patterns of YFP-ZRANB3 and YFP-PCNA expression in U2OS cells. (B) Interaction of ZRANB3 with PCNA is not affected by DNA damage. 293T cells were transiently transfected with empty vector or FLAG-tagged wild type ZRANB3, and where indicated exposed to UV irradiation. After 6 hours cells were lysed and subjected to

3 immunoprecipitation on anti-flag beads. Immunocomplexes were eluted by 3xFLAG peptide and analysed by Western blotting. Expression of FLAG-ZRANB3 is detectable only after immunoprecipitation. (C) Sensitivity of ZRANB3 deficient cells to DNA damaging agents. (D) GST-tagged wild type and mutant NZF proteins used in Fig. 2E. (E) Alignment of the HNH domains. Shown are representative HNH domains of ZRANB3 proteins, as well as free-standing bacterial HNH domains, and bacterial HNH domains found in Type III restriction endonucleases. Conserved residues are marked by asterisks, and the catalytic residue His121 is indicated by an arrow. (F) The HNH domain of ZRANB3 preferentially binds fork DNA, albeit with low affinity. Increasing concentrations of the HNH domain (residues of the full length ZRANB3) were incubated with the indicated 32 P-labelled DNA molecules and assayed by gel mobility shift analysis. Protein-DNA complexes are denoted by an asterisks *. (G) Full length ZRANB3 binds fork DNA with greater affinity than the isolated HNH domain. Increasing amounts of the full length ZRANB3 and the HNH domain (.5, 1.5 and 3 g) were incubated with 32 P-labelled fork DNA and assayed by gel mobility shift analysis. Protein-DNA complexes are denoted by an asterisks *. Supplemental Figure 3. (A) Purification of ZRANB3 protein on the size exclusion column. Peak fractions shown on the Coomassie stained gel (1 to 1; left) were assayed in the nuclease assay with the indicated fluorescently labelled DNA fork substrate (right). (B) Purified wild type ZRANB3, H121A and K65R and proteins. (C) ZRANB3 catalyses time-dependent conversion of a splayed DNA duplex into a faster migrating product. Splayed DNA duplex fluorescently labelled at 5 -end (shown in the picture), was incubated with ZRANB3 in the presence of ATP. Reactions were terminated at indicated times and analysed by native polyacrylamide gel electrophoresis. (D) Quantification of reactions shown in (C).

4 (E) ATPase assay in the presence of single-stranded, double stranded and fork DNA. ZRANB3 was incubated with 32 P-labelled ATP in the presence of indicated DNA substrates. Reaction products were resolved by thin layer chromatography. (F) Quantification of reactions shown in (E). Supplemental Figure 4. (A) Nuclease assay with the radiolabelled DNA substrate. 1 nm of 32 P-labelled splayed DNA duplex (indicated in the picture) was incubated with 2 nm of indicated enzymes in the presence or absence of ATP. Reactions were resolved by native polyacrylamide gel electrophoresis and analysed by autoradiography. (B) Nuclease assay with mutant ZRANB3 proteins. Wild type ZRANB3, the ATPase dead K65R mutant, the HNH mutant H121A, and the HNH mutant were incubated with a splayed DNA duplex fluorescently labelled at 5 -end (shown in the picture) in the presence of ATP. Reactions were analysed by native polyacrylamide gel electrophoresis. (C) Nuclease assay with the full length ZRANB3 proteins. Wild type ZRANB3, the ATPase dead K65R mutant, and the HNH mutant H121A were incubated with a splayed DNA duplex fluorescently labelled at 5 -end (shown in the picture) in the presence of ATP. Additionally, wild type ZRANB3 and FEN1 were incubated with the same substrate in the absence of ATP. Reactions were analysed by native polyacrylamide gel electrophoresis. (D) Ability of ZRANB3 to cleave different DNA substrates. Wild type ZRANB3 was incubated with the indicated fluorescently labelled DNA substrates in the presence of ATP. Reactions were analysed by native polyacrylamide gel electrophoresis. (E) ZRANB3 is unable to cleave splayed DNA duplexes with short DNA flaps. Wild type ZRANB3 was incubated with the indicated fluorescently labelled DNA substrates in the presence of ATP. Reactions were analysed by native polyacrylamide gel electrophoresis. (F) Ability of the NZF motif to interact with polyubiquitin chains is compared to the UBZ and UBM ubiquitin binding motifs of Pol and Pol, respectively. NZF, UBZ and UBM motifs were expressed as GST-fusion proteins and bound to the GST beads. The beads were then

5 incubated with polyubiquitin K48(2-7) or K63(2-7) chains. Interactions were assayed by antiubiquitin Western blotting. (G) Like ZRANB3, Polh and Polk colocalize with PCNA and ubiquitin conjugates. U2OS cells were transiently transfected with YFP-Pol or YFP-Pol, and immunostained with PCNA or FK2 antibodies. FK2 antibody recognizes ubiquitin conjugates, but not free ubiquitin. Supplemental Figure 5. (A) Overexpression of ZRANB3 does not induce H2AX and 53BP1 foci formation. U2OS cells were transfected with YFP-ZRANB3 and stained with rabbit 53BP1 antibody, or costained with mouse H2AX and rabbit PCNA antibodies. Anti-rabbit and anti-mouse Alexa Fluor 594 secondary antibodies were used to detect 53BP1 and H2AX, respectively, whereas anti-rabbit Alexa Fluor 647 was used to detect PCNA. (B) ZRANB3 accumulates at replication forks stalled at MMS and H 2 O 2 -induced DNA damage. U2OS cells were transfected with YFP-ZRANB3 and exposed to the indicated doses of MMS irradiation. After 6 h, cells were fixed and stained with PCNA antibody. They were analysed by microscopy, and percentages of cells containing ZRANB3 foci which colocalized with PCNA were determined. (C) Representative images of mutant ZRANB3 proteins which form foci that colocalize with PCNA. Note that the efficiency of colocalization with PCNA varies between different mutants, and is indicated in Fig. 5D. (D) Recruitment of mutant ZRANB3 proteins to laser-induced DNA damage. U2OS cells were transiently transfected with the indicated YFP-ZRANB3 constructs and analysed by live-cell imaging. Shown are representative images before, during and 2 min after induction of damage. Supplemental Figure 6. Synthetic oligonucleotides used in nuclease assays with ZRANB3. Shown DNA sequences correspond to the red DNA strands in the diagrams. Cy3 5 -end or FITC 3 -end labelled

6 oligonucleotides were annealed to the appropriate unlabelled oligonucleotides to obtain the DNA substrates used in nuclease assays.

7 Weston Supplemental Fig. 1. A B Replication associated DNA repair DNA damage tolerance Stalled replication fork Stalled replication fork Fork reversal Translesion synthesis Fork reversal Recombination-like Template switching Excision-based repair Resumption of DNA replicaiton

8 Weston Supplemental Fig. 2. A YFP- ZRANB3 B FLAG FLAG+UV Inputs ZRANB3 ZRANB3+UV FLAG FLAG+UV IPs ZRANB3 ZRANB3+UV αflag YFP-PCNA αγh2ax αpcna C Live cells % D plko shzranb nm CPT GST WT W625A T631A Y632A I633A N634A Live cells % E642A M643A mm HU E ZRANB3 HNH endonucl. Type III rest. enz. plko shzranb3-2 Live cells % plko shzranb3-1 shzranb µm H 2 O 2 * * * ** * ** * H.sapiens 961 EH-GVCQLCNVNAQELFLRLRDA [32] HFWQVDHIKPVYGGGGQCSLDNLQTLCTVCHKER M.musculus 951 EH-GVCQHCGVDAQELFLRMRDA [32] HFWQVDHIRPVYEGGGQCSLDNLQTLCTVCHKER T.guttata 98 EH-GVCQCCQHNAQELYLSVRDA [32] QFWQVDHIQPVYSGGGQCSLENLQTLCTACHRER A.thaliana 127 EH-GICTNCKLDCHQLVKRLRPL [33] NAWHADHIIPVYQGGGECRLENMRTLCVACHADV P.knowlesi 1394 DK-GVCNICKLDCTILIRQIKSR [36] HIWNVDHILPVFRGGGEASFDNLQTLCTFCHKKK Acidobacterium 84 DH-GLCALCQADTPAIYAALKRA [21] SLWDADHILPVAEGGGQCDLDNLRTLCLPCHREV S.usitatus 59 DK-GVCALCGVDTELLRKDKRKL [16] HLWDADHILPVAAGGGECGLANMRTLCLMCHRAR S.rotundus 25 DQ-ARCAWCGAP YAQVDHIVPVAFGGPEQDPDNMQCLCDVCHGAK C.jejuni 62 NKNPFCAKCGKFAK IIDHIVPIKQGGEKLSEENLQSLCIVCHNEK E.coli 23 SK-GICENCGKNAPFYLN [3] -PYLEVHHVIPLSSGGA-DTTDNCVALCPNCHREL A.variabilis 696 DS-YTCLCCGANTE AKLQVDHIKPFSMGG-ETSIQNSQTLCNICNKCK C.perfringens 687 EGLYYCNKCGYTSNF [1] -GMFQIDHIKPISKGG-LTTLDNLQLLCSKCNKIK D.radiodurans 731 DR--VCLCCGKRT QLQVDHIQSRYAGG-THDLDNLQLLCQVCNNLK Live cells % plko shzranb3-1 shzranb J/m 2 UV F DNA substrate HNH G HNH * *

9 Weston Supplemental Fig. 3. A Fractions: B H121A K65R C +ZRANB3 Time (min): D Cleaved DNA (%) Time (min) E - Enzyme - DNA ssdna dsdna 5 nm DNA 1 nm DNA 2 nm DNA 7 nm DNA Fork DNA ssdna dsdna Fork DNA ssdna dsdna Fork DNA ssdna dsdna Fork DNA F % ATP hydrolysed Fork DNA dsdna ssdna c (DNA)/nM

10 Weston Supplemental Fig. 4. ATP: A ssdna K65R H121A HNH FEN1 + + B K65R H121A HNH C ATP: H121A K65R FEN1 D ZRANB3: ssdna dsdna Fork DNA H121A WT H121A WT H121A WT E Flap length: ZRANB3: 24 nt 23 nt 2 nt 13 nt Marker DNA F K48-PolyUb GST GST-UBZ GST-UBM GST-NZF K63-PolyUb GST GST-UBZ GST-UBM GST-NZF G DAPI YFP-Polη αpcna Merge DAPI YFP-Polη αub conjug. Merge DAPI YFP-Polκ αpcna Merge αubiquitin DAPI YFP-Polκ αub conjug. Merge

11 Weston Supplemental Fig. 5. A DAPI YFP-ZRANB3 α53bp1 Merge YFP-ZRANB3 + 53BP1 DAPI YFP-ZRANB3 αγh2ax αpcna Merge Merge YFP-ZRANB3 YFP-ZRANB3 + PCNA + γh2ax B C DAPI YFP- αpcna Merge Cells % Cells % Cells containing foci Cells without foci mm MMS Cells containing foci Cells without foci mm H 2 O 2 DNA damage induced foci DNA damage induced foci NZF* PIP* PIP*NZF* HNH HNH HNH-NZF* D Before irradiation HNH HNH HNH-NZF* HNH-PIP* Photobleaching HNH- PIP*NZF* 2 min

12 Weston Supplemental Fig. 6. Splayed DNA duplex 5 -AGGTCTCGACTAACTCTAGTCGTTGTTCCACCCGTCCACCCGACGCCACCTCCTG-3 Cy3-5 -AGGTCTCGACTAACTCTAGTCGTTGTTCCACCCGTCCACCCGACGCCACCTCCTG-3 5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGATTGAAATTTAGGCTGGCACGGTCG-3 Cy3-5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGATTGAAATTTAGGCTGGCACGGTCG-3 5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGATTGAAATTTAGGCTGGCACGGTCG-3 -FITC dsdna 5 -CGACCGTGCCAGCCTAAATTTCAATCCACCCGTCCACCCGACGCCACCTCCTGC-3 3 Flap 5 -ACAACGACTAGAGTTAGTCGAGACCT-3 Splayed duplex with short 5 block 5 -CTAGAGTTAGTCGAGACCT-3 5 Flap 5 -CGACCGTGCCAGCCTAAATTTCAA-3 Splayed duplex with short 3 block 5 -CGACCGTGCCAGCCTAA-3 DNA bubble 5 -CGACCGTGCCAGCCATTTGGAGTTTCCACCCGTCCACCCGACGCCACCTCCTGC-3 DNA duplex with 3 overhang 5 -TCCACCCGTCCACCCGACGCCACCTCCTG-3 Four-way junction 5 -AGGTCTCGACTAACTCTAGTCGTTGTTCCACCCGTCCACCCGACGCCACCTCCTG-3 Cy3-5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGATTGAAATTTAGGCTGGCACGGTCG-3 5 -CAAGATTTTTACACGAATAGGGTGTAACAACGACTAGAGTTAGTCGAGACCT-3 5 -CGACCGTGCCAGCCTAAATTTCAATACACCCTATTCGTGTAAAAATCTTG-3 24 nt flap 5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGATTGAAATTTAGGCTGGCACGGTCG-3 23 nt flap 5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGAATGAAATTTAGGCTGGCACGGTCG-3 23 nt flap 5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGAACAAAATTTAGGCTGGCACGGTCG-3 13 nt flap 5 -GCAGGAGGTGGCGTCGGGTGGACGGGTGGAACAACGACTAGGCTGGCACGGTCG-3