SUPPLEMENTARY INFORMATION

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1 doi:1.138/nature1895 i Core histones CAF-1 v ii Reb1 CAF-1 Fen1 vi Mature Okazaki fragment iii PCNA Reb1 iv vii Mature Okazaki fragment Reb1 Mature Okazaki fragments Figure S1. Model for nucleosome-mediated dissociation and recycling of Pol δ. (i) Following Okazaki fragment synthesis, a (H3H4)2 tetramer is rapidly deposited on the fragment; (ii-iii) during elongation of the subsequent fragment, Pol δ and associated nucleases can invade this nascent tetramer (which may have matured via the addition of H2A/H2B into a complete nucleosome). (iv) The polymerase experiences resistance that increases as a function of proximity to the nucleosome dyad, and is accordingly most likely to dissociate around this point. Repeated cycles (v) of such dissociation produce fragments sized similarly to the chromatin repeat, with termini statistically enriched around nucleosome dyads. (vi-viii) The transcription factors Abf1, Reb1 and Rap1 present hard barriers that either cannot be invaded, or dissociate completely upon partial displacement by polymerase: these factors give rise to precisely located termini at a location corresponding to the edge of the transcription factor. 1

2 RESEARCH SUPPLEMENTARY INFORMATION a b c Length (bp) 25 C wt cdc9-1 CsCl gradient Fraction from Bottom RNase H + oligo - α-pol2 α-5s rrna Uncleaved >1, Genomic DNA Small RNA Cleaved Figure S2. Previously described 125 nt Okazaki fragments in S. cerevisiae are contaminating 5S rrna. (a) A 125 nt species previously described as Okazaki fragments with RNA primers still attached (43,44). Nucleic acids purified in the absence of RNase via CsCl density gradient centrifugation were labeled using Scriptcap capping enzyme (Cellscript) and α-gtp (specific for RNA 5 triphosphates). The 125nt species is present in both wild-type and cdc9-1 mutant cells, even at the permissive temperature. (b) Genomic DNA prepared using CsCl density gradient centrifugation contains large amounts of contaminating 5S rrna and trna. The genomic DNA migrates as a single band of high molecular weight in a native agarose gel. (c) The 125 nt species is 5S rrna. Labeled material was purified from the gel shown in Fig. S2C and subjected to RNase H treatment with oligonucleotides directed against either 5S rrna (lane 3) or a negative control RNA encoding DNA polymerase ε (Pol2 lane 2). Specific cleavage is observed only when an oligonucleotide directed against 5S rrna is used. 2

3 RESEARCH Dox-repressible Cdc9 Genotype 15 min Dox treatment WT lig4δ WT rad9δ tof1δ Figure S3. Okazaki fragment length is unaffected by checkpoint mutants, but abundance is increased. Asynchronous log-phase cultures (grown in YPD at 3 C) of the indicated strains were treated with doxycycline for 2.5h. DNA was purified and labeled as in Fig. 1a of main text. 3

4 RESEARCH SUPPLEMENTARY INFORMATION 1. Initiation 3. Ligation 2. Extension/ strand displacement 3 5 OH p 3 OH 5 p Purify OF-enriched DNA from 1-5 ml culture: denature at 95 C, Bind to Source 15Q at ph12, 3 mm NaCl Elute with increasing NaCl (5 mm step gradient) Ethanol precipitate fractions of interest (8-9 mm NaCl) [NaCl] M Input size (nt) mM Ligate preannealed sequencing adaptors directly to purified ssdna (T4 DNA ligase, overnight incubation at 16 C) ` 5 5 p NNNNNN 5 Illumina Truseq primer 1 with 5 N6 ss overhang 3 3 OH p NNNNNN 3 Illumina Truseq primer 2 with 3 N6 ss overhang Synthesise second strand (Taq DNA polymerase) Purify from native 2.5% agarose gel (2-1bp) Amplify by PCR (16 cycles, KOD hot start) Purify from two sequential 2.5% agarose gels (2-6bp) ` Figure S4. Strategy for Okazaki fragment purification and library amplification. Workflow for Okazaki fragment purification and deep-sequencing library generation. Full details are in supplemental methods. 4

5 RESEARCH Origin WT dataset 1 Confirmed origins Watson Crick Watson strand hits 2 Log2 watson:crick 1-1 Crick strand hits Position relative to ACS (bp) (252 ACS from Eaton et al., 21) WT dataset 2 Watson strand hits 2 Log2 watson:crick 1-1 Crick strand hits Position relative to ACS (bp) (252 ACS from Eaton et al., 21) pol32δ dataset 1 Watson strand hits 2 Log2 watson:crick 1-1 Crick strand hits Position relative to ACS (bp) (252 ACS from Eaton et al., 21) pol32δ dataset 2 Watson strand hits 2 Log2 watson:crick 1-1 Crick strand hits Position relative to ACS (bp) (252 ACS from Eaton et al., 21) Figure S5. Global distribution and strand bias of Okazaki fragments is highly reproducible across wild-type and pol32δ strains. As in Fig. 2a of main text, the distribution of paired-end sequencing hits mapping to either the Watson (blue, above the axis) or Crick (orange, below) strands across S. cerevisiae chromosome 1. Replicate samples of both wild-type and pol32δ strains are shown. The locations of origins confirmed by Sekedat et al (45) are indicated. Data are unsmoothed. Meta-analysis of strand bias around active ACS sites, calculated as the log2 ratio of Watson strand to Crick strand Okazaki fragment 3 ends over a 5 kb window around the ACS sequences of active origins (46), is shown beside each trace. Data in this analysis are smoothed to 2bp. 5

6 RESEARCH SUPPLEMENTARY INFORMATION a Length distribution of sequenced fragments (wt) b Length distribution of sequenced fragments, log scale (wt) 1 8 Dataset 1 [Figs 2-4, S6C(ii)] Dataset 2: [Fig S6C(ii)] 1 8 Dataset 1 [Figs 2-4, S6C(ii)] Dataset 2: [Fig S6C(ii)] Count 6 4 Monosome sample Disome subset Count 6 4 Monosome sample Disome subset Length (nt) 1 1 Length (nt) c (i) WT dataset 1 All fragments over 1nt Monosome-sized Okazaki fragments Normal distribution: Mean=165, SD=15 Dinucleosome-sized Okazaki fragments (All fragments from nt) Relative Count Nucleosome occupancy (Kaplan et al. 29) Position relative to nucleosome dyad (bp) (28832 best consensus centres; Jiang and Pugh, 29) (ii) WT dataset 2 All fragments over 1nt Monosome-sized Okazaki fragments Normal distribution: Mean=165, SD=15 Dinucleosome-sized Okazaki fragments (All fragments from nt) Relative Count Nucleosome occupancy (Kaplan et al. 29) Position relative to nucleosome dyad (bp) (28832 best consensus centres; Jiang and Pugh, 29) Figure S6. Length distributions of sequenced Okazaki fragments, and relationship to nucleosome locations, from a wild-type strain. (a) The length distribution of sequenced fragments from dataset 1 (grey) or dataset 2 (black) wild-type samples is shown; fragments under 1 nt (shaded area) are discarded. (b) As (a) but with logarithmic x-axis scale A comparison between the length profiles of labeled and sequenced fragments is shown, together with schematic representations of the data used to generate the monosome-sized samples and disome-sized subsets shown in Fig. 3b and S6c. We note that, because labeling occurs at DNA ends, the intensity of signal on the autoradiogram is directly proportional to fragment number regardless of length. The monosome-sized subsets were generated by selecting fragments at random to conform to an integer approximation of the normal distribution with mean 165 nt and standard deviation 15 nt. The resulting subsets contain 1,851,13 unique combinations of 5 and 3 ends. Due to lower count numbers at increased lengths, disome-sized fragments were more simply defined as all fragments from nt. (c) Comparison of the distribution of Okazaki fragment termini about nucleosome midpoints for (i) dataset 1 and (ii) dataset 2 samples. The distributions of all fragments (left), the monosome-size sample shown in Fig. 3b (middle), and dinucleosome-sized fragments (right) are shown. Data are unsmoothed and normalized to the maximum signal in the analysed range. 6

7 RESEARCH a Location of mature 3 and 5 ends Flap RNA primer (Degraded) Fork b c TSS TSS Relative Count Nucleosome occupancy (Whitehouse et al., 27) occupancy (MacIsaac et al., 26) Long : Short Log ratio Position relative to TSS (bp) Position relative to TSS (bp) Figure S7. The distribution of fragments synthesized in the opposite direction to transcription around TSS supports the model for Pol δ recycling. (a) Schematic representation of the relative orientations of transcription start sites and Okazaki fragments used in this analysis. Note that, unlike in other figures, Okazaki fragment synthesis proceeds from right to left. (b) The distribution of Okazaki fragment termini correlates with nucleosome occupancy around transcription start sites. Data are aligned to TSS such that the direction of transcription is from left to right: only Okazaki fragments synthesized in the opposite direction are plotted here this is a companion analysis to that shown in Fig. 3 c. Data are smoothed to 5bp and normalized to the maximum signal in the analysed range. (c) Companion analysis to that shown in Fig. 3 d. Okazaki fragments synthesized against transcription, and whose ends flank nucleosome-depleted regions are disproportionately long. Equal numbers of reads were selected from the top (long), and the bottom three (short) length quartiles of sequenced fragments. The log2 ratio of long to short fragments is plotted. Data are smoothed to 5bp. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Model 1: Model 2: Bulk nucleosomes Bulk nucleosomes C M G Pol α PCNA Fen1 C M G Pol α PCNA Fen1 Chromatin is rapidly reassembled behind the fork Pol δ invades nascent nucleosomes, which facilitate its dissociation Fixed distance from nucleosome to initiation site Constant amount of strand displacement synthesis by Pol δ Nicks at nucleosome dyads Position determined by nucleosome at site of nick NDRs Nucleosome dyad directing nick location Nicks at nucleosome dyads Position determined by nucleosome upstream of nick NDRs Nucleosome edge directing nick location Relative probability of Pol δ dissociation Relative probability of Pol δ dissociation Predicted observations: Nicks at dyads of both +1 and -1 nucleosomes Longest fragments span the NFR from -1 dyad to +1 dyad Nicks enriched on replication-fork-proximal side of transcription factor binding sites Observed? Fig. 3C Fig. 3D Fig. 3C, 4 Predicted observations: Few nicks at dyad of +1 nucleosome Longest fragments from mid-nfr to +2 dyad Nicks offset from transcription factor binding sites Observed? Fig. 3C Fig. 3D Fig. 3C, 4 Figure S8. Competing models that could give rise to the observed size and location of Okazaki fragments in bulk chromatin, and predictions for their behavior around nucleosome-depleted regions. Schematic representation of two models that could explain the observed size and distribution of Okazaki fragments: model 1 corresponds to the model shown in Fig. S1. The predicted behaviour around nucleosome-depleted regions differs, and is shown for each model together with an indication of whether this behavior is observed. Model 1. Chromatin is reassembled extremely rapidly behind the replication fork; during strand displacement synthesis, Pol δ invades nascent nucleosomes, experiencing increased resistance as it approaches the dyad. Dissociation is most likely around the middle of the nucleosome, and the nick corresponding to Okazaki fragment termini lies within the nucleosome that caused dissociation. Model 2. Okazaki fragment initiation by Pol α is biased by collisions between the replisome and nucleosomes ahead of the replication fork. Due to the large footprint of the Cdc45/Mcm2-7/GINS (CMG) replicative helicase and Pol α itself, initiation occurs a fixed distance of ~6 bp from the edge of the nucleosome ahead of the fork: the site of the nick is moved by Pol δ, and the extent of strand displacement is ~3 bp. The nick is therefore ultimately located ~9bp from the edge of the upstream nucleosome; in bulk chromatin with a ~2bp linker region, this site corresponds to the dyad of the downstream nucleosome. 8

9 RESEARCH a WT dataset 1 All TF binding sites All excluding only High-resolution Relative count b pol32δ dataset 1 All TF binding sites All excluding only High-resolution Relative count c WT dataset 2 All TF binding sites All excluding only High-resolution Relative count Relative count d pol32δ dataset 2 All TF binding sites All excluding only High-resolution Figure S9. Enrichment of Okazaki fragment termini around Abf1, Reb1 and Rap1 binding sites is highly reproducible across wild-type and pol32δ strains. The distribution of Okazaki fragment termini around transcription factor binding sites (including, excluding or limited to Abf1, Reb1 and Rap1 sites) is shown for (a) wild-type, (b) pol32δ, and (c-d) respective replicate samples. Data are unsmoothed and normalized to the maximum signal in the analysed range. 9

10 RESEARCH SUPPLEMENTARY INFORMATION a TSS TSS TF Pol TF Pol Fork Fork TF Pol TF Pol TF Pol TF Pol b trna genes centred on TSS Fragment synthesis trna genes centred on TSS Fragment synthesis Hits per bp (5bp smoothed) Position relative to TSS (bp) Transcribed region TFIIIB binding site Position relative to TSS (bp) Hits per bp (5bp smoothed) Figure S1. Distribution of Okazaki fragment termini around trna genes. (a) Schematic representation of the expected interaction between transcription factors, RNA polymerase, and the lagging strand polymerase: while interaction with transcription factors is anticipated for fragments synthesized in either direction relative to transcription, an interaction with RNA polymerase should be observed only when fragments are synthesized against the direction of transcription, requiring polymerase to transcribe from an Okazaki fragment template. (b) Distribution of Okazaki fragment termini around trna genes, separated by direction of synthesis as indicated. Increased density is observed on the side of the TFIIIB binding site first encountered by Pol δ as predicted by our model. In addition, a large increase in end density is observed at a site predicted to correspond to the location of an RNA polymerase stalled at the 3 end of the gene. 1

11 RESEARCH a Length distribution of sequenced fragments (pol32δ) Length distribution of sequenced fragments, log scale (pol32δ) Count Dataset 1 [Fig. 5C, S11C(ii)] Dataset 2 [Fig. S11C(ii)] Monosome sample Disome subset Count Dataset 1 [Fig. 5C, S11C(ii)] Dataset 2 [Fig. S11C(ii)] Monosome sample Disome subset Length (nt) 1 1 Length (nt) b (i) pol32δ dataset 1 All fragments over 1nt Monosome-sized Okazaki fragments Normal distribution: Mean=165, SD=15 Dinucleosome-sized Okazaki fragments (All fragments from nt) Relative Count Nucleosome occupancy (Kaplan et al. 29) Position relative to nucleosome dyad (bp) (28832 best consensus centres; Jiang and Pugh, 29) (ii) pol32δ dataset 2 All fragments over 1nt Monosome-sized Okazaki fragments Normal distribution: Mean=165, SD=15 Dinucleosome-sized Okazaki fragments (All fragments from nt) Relative Count Nucleosome occupancy (Kaplan et al. 29) Position relative to nucleosome dyad (bp) (28832 best consensus centres; Jiang and Pugh, 29) Figure S11. Length distributions of sequenced Okazaki fragments, and relationship to nucleosome locations, from a pol32δ strain. (a) The length distributions of sequenced fragments from dataset 1(grey) or dataset 2 (black) pol32δ samples is shown with linear (left) and logarithmic (right) x-axis scales, together with schematic representations of the monosome-sized samples and disome-sized subsets shown in Fig. 5c and S11b. (b) Comparison of the distribution of Okazaki fragment termini about nucleosome midpoints for (i) experimental and (ii) replicate samples. The distributions of all fragments (left), the monosomesize sample shown in Fig. 5c (middle), and dinucleosome-sized fragments (right) are shown. Data are unsmoothed and normalized to the maximum signal in the analysed range. 11

12 RESEARCH SUPPLEMENTARY INFORMATION Supplemental tables Table S1. Strains used in this study * denotes strain used for Okazaki fragment deep sequencing yiw5 MATa; his3-1; met15-; ; ubr1::ppgk1-ubr1 (LEU2); cdc9::pcmv-tetr-vp16 ptetr-ubi4-cdc9 (URA3) yiw53 MATa; his3-1; met15-; rad9::kanmx; ubr1::ppgk1-ubr1 (LEU2); cdc9::pcmv-tetr-vp16 ptetr-ubi4- CDC9 (URA3) yiw121 MATa; his3-1; met15-; dnl4::hyg; tof1::kanmx; ubr1::pgal1-ubr1 (LEU2); cdc9::pcmv-tetr- VP16 ptetr-ubi4-cdc9 (URA3) yiw16* MATa; his3-1; met15-; dnl4::hyg; rad9::kanmx; ubr1::pgal1-ubr1 (LEU2); cdc9::pcmv-tetr- VP16 ptetr-ubi4-cdc9 (URA3) yiw174* MATa; his3-1; met15-; dnl4::hyg; rad9::kanmx; pol32::nat ubr1::pgal1-ubr1 (LEU2); cdc9::pcmv- TETR-VP16 ptetr-ubi4-cdc9 (URA3) yiw193 MATa; his3-1; met15-; dnl4::hyg; rad9::kanmx; cac1::nat ubr1::pgal1-ubr1 (LEU2); cdc9::pcmv- TETR-VP16 ptetr-ubi4-cdc9 (URA3) yiw24 MATa; his3-1; met15-; dnl4::hyg; rad9::kanmx; cac2::nat ubr1::pgal1-ubr1 (LEU2); cdc9::pcmv- TETR-VP16 ptetr-ubi4-cdc9 (URA3) yiw25 MATa; his3-1; met15-; dnl4::hyg; rad9::kanmx; cac3::nat ubr1::pgal1-ubr1 (LEU2); cdc9::pcmv- TETR-VP16 ptetr-ubi4-cdc9 (URA3) 12

13 RESEARCH Supplementary References 43. Bielinsky, A. K. & Gerbi, S. A. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279, (1998) Bielinsky, A. K. & Gerbi, S. A. Chromosomal ARS1 has a single leading strand start site. Mol Cell 3, (1999). 45. Sekedat, M. D. et al. GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome. Mol Syst Biol 6, 353 (21). 46. Eaton, M. L., Galani, K., Kang, S., Bell, S. P. & MacAlpine, D. M. Conserved nucleosome positioning defines replication origins. Genes Dev 24, (21). 13