Acknowledgments: - Working towards understanding DNA replication : coupling of a 3'-5' helicase

and the nucleoprotein complex formed quantified by phosphorimaging.

4.7.11 ATPase assay.

Reactions (20µl) containing 25 mM Hepes-NaOH/pH7.5, 5 mM NaCl, 10 mM magnesium acetate, 1 mM DTT, 0.1 mg/ml BSA, 66 nM [α-³²P] ATP (3,000 Ci/mmol), and various levels of unlabeled ATP were incubated at 37^◦C for 30 min. Aliquots (1µl) were spotted on a polyethyleneimine-cellulose TLC plate (Merck) and developed in 0.5 M LiCl/1.0 M formic acid and the products analyzed by Phosphorimaging.

4.7.12 Preparation of 200-nt primed circle.

A 200-nt minicircle DNA substrate (containing only C, A and G) was prepared as follows:

two reactions (30µl) each containing 1 nmol of the 100-nt oligonucleotides A or B (se-quences in Table S1) were incubated with T4 polynucleotide kinase (40 U, New England Biolabs) and [γ-³²P]-ATP (1 mM, 350 cpm/pmol) for 1 h at 37^◦C. The phosphorylated oligonucleotides A and B were combined, mixed with 5 nmol of the oligonucleotide Bridges AB and BA in 20 mM Hepes-NaOH/pH7.5 and 150 mM NaCl in a total volume of 100µl and the mixture heated to 100^◦C for 10 min and then cooled slowly to 22^◦C. Ligation of the oligonucleotides was carried out in reactions (525µl) containing T4 DNA ligase (8000 U), T4 DNA ligase buffer (New England Biolabs) and 1 mM AT and 16^◦C for 14 h.

The ligation was monitored with Exol and ExoIII (New England Biolabs). Following complete ligation, the products were subjected to 8 M urea- 10% PAGE separation in 1x TBE at 15 W for 75 min; the gel region containing the circular 200-nt ssDNA was excised and the DNA eluted with 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA. After ethanol precipitation, the circular 200-nt ssDNA was resuspended in 30µl of 1xTE/pH 8.0 and the solution passed through a S300 mini- column filter (GE Health-care). The procedure yielded -20% of the input oligonucleotide as the circular 200-nt ssDNA.

To obtain the rolling circle substrate 9 pmol of the minicircle was annealed to 9 pmol of oligonucleotide C 100^◦C, boiling for 3 minutes and slowly cooling it to 22^◦C; the mixture was applied on a S300 minicolumn (GE Healthcare) and eluted in 1x TE. The concentration of substrate was assessed by UV absorption.

4.8 Acknowledgments:

This study was supported by the National Institute of Health Grant GMS R01 GM034559.

Figure 1.3 DNA helicase activity of the hCMG complex on various DNA substrates. (A) Increasing levels of CMG (7.5, 15, and 30 fmol) were incubated with oligonucleotide substrates (see table S1 for description of oligonucleotides used in this assay). The first two substrates (I and II), shown in the upper panel of the figure, were formed by annealing oligonucleotide #3 (labeled) with #9 or #11, respectively. Substrate III was prepared by annealing oligonucleotide #3 (labeled) with #11 (bottom strand) and #14. Substrate IV was made by annealing oligonucleotide #15 (labeled) with #5 and #9 (bottom strand). The three substrates (V, VI, and VII), shown in lower panel of the figure, were synthesized by annealing oligonucleotide #3 (labeled) with oligonucleotide #12, #13 or #10, respectively. Substrate VIII was made by annealing oligonucleotide #3 (labeled) with oligonucleotides #12 (bottom strand) and #14. Substrate IX was generated by annealing oligonucleotide #14 (labeled) with oligonucleotides #12 (bottom strand) and #3. (B) Helicase assays with oligonucleotide substrates containing different 3’ tail lengths. Substrates containing the 3’-oligo dT₄₀ or 3'-oligo dT80 tails are the same as I and VII described in panel A. Substrates containing 3’-oligo dT20 tails was made by annealing oligonucleotide #3 (labeled) with #8. CMG (15 fmol) was incubated with substrates for varying incubation periods and the substrate unwound (%) plotted against the time of incubation. (C) Helicase assays with M13 substrates containing a 39-mer duplex region and different 5’-dT tail lengths (0, 20-, 40-, and 60-nt) prepared by annealing labeled oligonucleotides #1, 2, 3, or 4 with M13. The unwound substrate formed (%) was calculated and plotted against the level of CMG complex added.

Fig. 4.3: DNA helicase activity of the hCMG complex on various DNA substrates.

(A) Increasing levels of CMG (7.5, 15, and 30 fmol) were incubated with oligonucleotide sub-strates (see tab. 4.1 for description of oligonucleotides used in this assay). The first two subsub-strates (I and II), shown in the upper panel of the figure, were formed by annealing oligonucleotide #3 (labeled) with #9 or #11, respectively. Substrate III was prepared by annealing oligonucleotide

#3 (labeled) with #11 (bottom strand) and #14. Substrate IV was made by annealing oligonu-cleotide #15 (labeled) with #5 and #9 (bottom strand). The three substrates (V, VI, and VII), shown in lower panel of the figure, were synthesized by annealing oligonucleotide #3 (labeled) with oligonucleotide #12, #13 or #10, respectively. Substrate VIII was made by annealing oligonucleotide #3 (labeled) with oligonucleotides #12 (bottom strand) and #14. Substrate IX was generated by annealing oligonucleotide #14 (labeled) with oligonucleotides #12 (bot-tom strand) and #3. (B) Helicase assays with oligonucleotide substrates containing different 3’ tail lengths. Substrates containing the 3’-oligo dT40 or 3’- oligo dT80 tails are the same as I and VII described in panel A. Substrates containing 3’-oligo dT20 tails was made by annealing oligonucleotide #3 (labeled) with #8. CMG (15 fmol) was incubated with substrates for varying incubation periods and the substrate unwound (%) plotted against the time of incubation. (C) Helicase assays with M13 substrates containing a 39-mer duplex region and different 5’-dT tail lengths (0, 20-, 40-, and 60-nt) prepared by annealing labeled oligonucleotides #1, 2, 3, or 4 with M13. The unwound substrate formed (%) was calculated and plotted against the level of CMG complex added.

4.8. Acknowledgments: 79

11 Figure 1.4 Processivity of the CMG helicase activity. (A) Substrates used for the processivity studies were prepared as described in “Materials and Methods”. For the preparation of the short duplex DNA substrate (39-500 bp) used in lanes 1-5, the annealed oligonucleotide was extended in the presence of dNTPs containing ddCTP. For the preparation of the long duplex substrate (>500 bp), described in lanes 6-10, the annealed oligonucleotide was extended in the absence of ddCTP. Increasing levels of CMG (12.5, 25, and 50 fmol) were incubated with DNA substrates as described in “Materials and Methods”. (B) Comparison of the processivity in the presence and absence of RPA or E. coli SSB. Substrates containing duplex regions, as shown, were pre-incubated with 40 fmol the hCMG complex in the presence of 0.05 mM ATP and then supplemented with 0.45 mM ATP and RPA (1.7 pmol) or E. coli SSB (0.27 pmol) followed by further incubation for 30 min.

Fig. 4.4: Processivity of the CMG helicase activity. (A) Substrates used for the processivity studies were prepared as described in ”Materials and Methods”. For the preparation of the short duplex DNA substrate (39-500 bp) used in lanes 1-5, the annealed oligonucleotide was extended in the presence of dNTPs containing ddCTP. For the preparation of the long duplex substrate (>500 bp), described in lanes 6-10, the annealed oligonucleotide was extended in the absence of ddCTP. Increasing levels of CMG (12.5, 25, and 50 fmol) were incubated with DNA substrates as described in ”Materials and Methods”. (B) Comparison of the processivity in the presence and absence of RPA or E. coli SSB. Substrates containing duplex regions, as shown, were pre-incubated with 40 fmol the hCMG complex in the presence of 0.05 mM ATP and then supplemented with 0.45 mM ATP and RPA (1.7 pmol) orE. coli SSB (0.27 pmol) followed by further incubation for 30 min.

Figure 1.5 DNA binding activity of the hCMG complex. EMSA assays were performed with DNA substrates under different conditions. Reactions contained 15 fmol of hCMG. (A) EMSA assays with fork-structured DNAs containing 5’- or 3’-oligo dT40 tails prepared by annealing oligonucleotides #3 and #9 (Table S1). In the right panel, EMSA assays were performed with the nucleotides indicated at the top of the gel. The DNA products formed (substrate-protein complex, substrate, and unwound product) are illustrated on the right side of A. (B) EMSA assays with substrates shown at top of gels. The first three substrates used were prepared by annealing oligonucleotides #1 with #7, #3 with #7, and #1 with #9. The next two substrates were prepared with labeled oligonucleotides #3 and #9 while the last substrate indicated was identical to that described in panel A. (C) CMG binding to fork-structured substrates containing different 3’

tail lengths. The substrates used were the same as those described in Fig. 3B, right panel. The nucleoprotein complex formed (%) was calculated and plotted against the CMG complex added.

Fig. 4.5: DNA binding activity of the hCMG complex EMSA assays were performed with DNA substrates under different conditions. Reactions contained 15 fmol of hCMG. (A) EMSA assays with fork- structured DNAs containing 5’- or 3’-oligo dT40 tails prepared by an-nealing oligonucleotides #3 and #9 (tab. 4.1). In the right panel, EMSA assays were performed with the nucleotides indicated at the top of the gel. The DNA products formed (substrate-protein complex, substrate, and unwound product) are illustrated on the right side of A. (B) EMSA assays with substrates shown at top of gels. The first three substrates used were prepared by annealing oligonucleotides #1 with #7, #3 with #7, and #1 with #9. The next two sub-strates were prepared with labeled oligonucleotides #3 and #9 while the last substrate indicated was identical to that described in panel A.(C)CMG binding to fork-structured substrates con-taining different 3’ tail lengths. The substrates used were the same as those described in fig. 4.3 (B), right panel. The nucleoprotein complex formed (%) was calculated and plotted against the CMG complex added.

4.8. Acknowledgments: 81

13 Figure 1.6 Rolling circle assay. The primed 200-nt mini-circle was synthesized as described in the supporting information. (A) Requirements for Pol !-catalized leading strand synthesis. Reactions (15 µl), containing 20 mM Tris-HCl/pH7.5, 10 mM magnesium acetate, 10 mM potassium glutamate, 1 mM DTT, 0.1 mg/ml BSA, 0.2 mM EDTA, 3.75% glycerol, 0.5 mM AMP-PNP, rolling circle DNA substrate (50 fmol) and hCMG complex (15 fmol) were incubated for 10 min at 37ºC; 0.12mM dCTP, 0.12 mM, dGTP, and 0.03 mM ["-³²P]-dATP (specific activity 37,700 cpm/pmol) were added with RFC (20 fmol), PCNA (1 pmol) and hPol ! (70 fmol). After 5 min at 37ºC, 5 mM ATP was added and the reaction incubated for 10 min after which E. coli SSB (0.5 pmol) was added and the mixture incubated for 60 min. Mixtures were adjusted to 10 mM EDTA and separated on an alkaline agarose gel (1%) at 15 W for 2.5 hr. The gel was washed with water, dried and audioradiographed at -80ºC. (B) Comparison of leading strand synthesis by hPol ! and hPol #. Reactions were as described in A with 50 mM potassium glutamate, 50 fmol CMG complex and 35 and 70 fmol hPol ! or 35, 70 and 300 fmol of hPol #, where indicated. After incubation, samples were treated with proteinase K (0.1 mg/ml) in reactions containing 20 mM EDTA, 1% SDS and 40 µg of yeast t-RNA.

Following ethanol precipitation, samples were subjected to alkaline agarose gel separation as described above. (C) Elongation of singly primed M13 by hPol ! and hPol #. The activity of hPol ! (55 fmol) and hPol

# (44 fmol) observed with singly primed M13 (7 fmol) following incubation for 30 min at 37ºC was carried out as previously described [17].

Fig. 4.6: Rolling circle assay. The primed 200-nt mini-circle was synthesized as described in the supporting information. (A) Requirements for Pol$-catalized leading strand syn-thesis. Reactions (15µl), containing 20 mM Tris-HCl/pH7.5, 10 mM magnesium acetate, 10 mM potassium glutamate, 1 mM DTT, 0.1 mg/ml BSA, 0.2 mM EDTA, 3.75% glycerol, 0.5 mM AMP-PNP, rolling circle DNA substrate (50 fmol) and hCMG complex (15 fmol) were incubated for 10 min at 37^◦C; 0.12 mM dCTP, 0.12 mM, dGTP, and 0.03 mM [α-³²P]-dATP (specific activity 37,700 cpm/pmol) were added with RFC (20 fmol), PCNA (1 pmol) and hPol$(70 fmol). After 5 min at 37^◦C, 5 mM ATP was added and the reaction incubated for 10 min after which E. coli SSB (0.5 pmol) was added and the mixture incubated for 60 min. Mixtures were adjusted to 10 mM EDTA and separated on an alkaline agarose gel (1%) at 15 W for 2.5 hr. The gel was washed with water, dried and audioradiographed at −80^◦C. (B) Comparison of leading strand synthesis by hPol $ and hPol δ. Reactions were as described in A with 50 mM potassium glutamate, 50 fmol CMG complex and 35 and 70 fmol hPol$or 35, 70 and 300 fmol of hPolδ, where indicated. After incubation, samples were treated with proteinase K (0.1 mg/ml) in reactions containing 20 mM EDTA, 1% SDS and 40µg of yeast t-RNA. Following ethanol precipitation, samples were subjected to alkaline agarose gel separation as described above. (C) Elongation of singly primed M13 by hPol$ and hPol δ. The activity of hPol$(55 fmol) and hPolδ (44 fmol) observed with singly primed M13 (7 fmol) following incubation for 30 min at 37^◦C was carried out as previously described [133].

17 1.6 Supplemental Figures

Supplementary Figure 1-1 Isolation of hCMG complex from 293 cells Western blot analysis of glycerol gradient fractions (5 µl) detected Mcm2, Cdc45 and Psf3 in the peak glycerol gradient fraction (#4) that contained helicase activity (determined with 2 µl aliquots). This fraction cosedimented with the thyroglobulin marker.

Supplementary Figure 1-2 Western blot analysis of the purified hCMG complex isolated from Sf9 cells.

Western blots against the components of the hCMG complex were carried out with glycerol gradient fractions (5µl) loaded onto 4-20% gel as described in Fig. 2A. The Mcm5 western blot was carried out on a membrane blotted previously with Mcm7.

Fig. 4.7: Isolation of hCMG complex from 293 cells. Western blot analysis of glycerol gradient fractions (5µl) detected Mcm2, Cdc45 and Psf3 in the peak glycerol gradient fraction (#4) that contained helicase activity (determined with 2µl aliquots). This fraction cosedimented with the thyroglobulin marker.

1.6 Supplemental Figures

Supplementary Figure 1-1 Isolation of hCMG complex from 293 cells Western blot analysis of glycerol gradient fractions (5 µl) detected Mcm2, Cdc45 and Psf3 in the peak glycerol gradient fraction (#4) that contained helicase activity (determined with 2 µl aliquots). This fraction cosedimented with the thyroglobulin marker.

Supplementary Figure 1-2 Western blot analysis of the purified hCMG complex isolated from Sf9 cells.

Fig. 4.8: Western blot analysis of the purified hCMG complex isolated from Sf9 cells. Western blots against the components of the hCMG complex were carried out with glycerol gradient fractions (5µl) loaded onto 4-20% gel as described in fig. 4.2(A). The Mcm5 western blot was carried out on a membrane blotted previously with Mcm7.

4.8. Acknowledgments: 83

Supplementary Figure 1-3 ATPase activity of the hCMG complex. ATPase activity of the hCMG complex was measured as described in “Materials and Methods” in the supporting information. The hCMG complex (15 fmol) was incubated with various levels of ATP (0.05, 0.2, 0.5, 1.5, and 4 mM) and the amount of ADP produced (molecules per min per molecule enzyme) was determined and plotted against the concentration of ATP added.

Supplementary Figure 1-4 Influence of ATP on the helicase activities of the hCMG and Mcm4/6/7 complexes using M13 substrates. (A) The helicase activity observed with hCMG (20 fmol) and (B) hMcm4/6/7 (200 fmol) complexes in the presence of various ATP levels. The amount of substrate unwound (%) was determined and plotted against the concentration of ATP added. The helicase substrate used in this experiment was prepared by annealing oligonucleotide #6 (labeled) with M13.

Fig. 4.9: ATPase activity of the hCMG complex. ATPase activity of the hCMG complex was measured as described in ”Materials and Methods” in the supporting information.

The hCMG complex (15 fmol) was incubated with various levels of ATP (0.05, 0.2, 0.5, 1.5, and 4 mM) and the amount of ADP produced (molecules per min per molecule enzyme) was determined and plotted against the concentration of ATP added.

18 Supplementary Figure 1-3 ATPase activity of the hCMG complex. ATPase activity of the hCMG complex was measured as described in “Materials and Methods” in the supporting information. The hCMG complex (15 fmol) was incubated with various levels of ATP (0.05, 0.2, 0.5, 1.5, and 4 mM) and the amount of ADP produced (molecules per min per molecule enzyme) was determined and plotted against the concentration of ATP added.

Fig. 4.10: Influence of ATP on the helicase activities of the hCMG and Mcm4/6/7 complexes using M13 substrates. (A)The helicase activity observed with hCMG (20 fmol) and (B)hMcm4/6/7 (200 fmol) complexes in the presence of various ATP levels. The amount of substrate unwound (%) was determined and plotted against the concentration of ATP added.

The helicase substrate used in this experiment was prepared by annealing oligonucleotide #6 (labeled) with M13.

19 Supplementary Figure 1-5 Comparison of the helicase activity of the hCMG and hMcm2-7 complexes.

CMG (20 fmol) and Mcm2-7 (25 – 200 fmol) complexes were assayed using standard helicase conditions (hCMG reaction contained 7.5 mM NaCl and the Mcm2-7 reaction contained 7.5 mM potassium acetate).

Experiments with the Mcm2-7 complex included the indicated levels of sodium acetate or sodium glutamate. Substrate were prepared by annealing oligonucleotides #3 (labeled) with #10.

Supplementary Figure 1-6 Influence of the 3’ tail sequence on hCMG helicase activity. The CMG helicase (15 fmol) activity was examined with substrates possessing 3'-tailed homopolymers (dT20, dA20, and dC20) and a 3'-tail containing a random sequence. The amount of substrate unwound (%) was determined and plotted against the incubation time. Substrates were prepared using labeled oligonucleotide #18 (upper strand) annealed to either oligonucelotides #8 (3’ dT20), #19 (3’ dA20), #20 (3’ dC20), or #21 (3’ random).

Fig. 4.11: Comparison of the helicase activity of the hCMG and hMcm2-7 com-plexes. CMG (20 fmol) and Mcm2-7 (25 - 200 fmol) complexes were assayed using standard helicase conditions (hCMG reaction contained 7.5 mM NaCl and the Mcm2-7 reaction contained 7.5 mM potassium acetate). Experiments with the Mcm2-7 complex included the indicated levels of sodium acetate or sodium glutamate. Substrate were prepared by annealing oligonucleotides

#3 (labeled) with #10.

19 Supplementary Figure 1-5 Comparison of the helicase activity of the hCMG and hMcm2-7 complexes.

CMG (20 fmol) and Mcm2-7 (25 – 200 fmol) complexes were assayed using standard helicase conditions (hCMG reaction contained 7.5 mM NaCl and the Mcm2-7 reaction contained 7.5 mM potassium acetate).

Experiments with the Mcm2-7 complex included the indicated levels of sodium acetate or sodium glutamate. Substrate were prepared by annealing oligonucleotides #3 (labeled) with #10.

Fig. 4.12: Influence of the 3’ tail sequence on hCMG helicase activity. The CMG he-licase (15 fmol) activity was examined with substrates possessing 3’-tailed homopolymers (dT₂₀, dA₂₀, and dC₂₀) and a 3’-tail containing a random sequence. The amount of substrate unwound (%) was determined and plotted against the incubation time. Substrates were prepared using labeled oligonucleotide #18 (upper strand) annealed to either oligonucelotides #8 (3’ dT₂₀),

#19 (3’ dA₂₀), #20 (3’ dC₂₀), or #21 (3’ random).

4.8. Acknowledgments: 85

Supplementary Figure 1-7 Influence of ATP levels on the hCMG helicase activity and its binding to oligonucleotide substrates. The helicase and DNA binding activities of the hCMG complex (20 fmol) were measured in the presence of different ATP levels. The amount of substrate unwound (%) and bound (%) were determined and plotted against the level of ATP added. The substrate was prepared by annealing oligonucleotides #16 (labeled) with #17.

Supplementary Figure 1-8 Purification of Mcm2-7 and Mcm4/6/7 complex. The Mcm2-7 and Mcm4/6/7 complexes were purified as described in “Materials and Methods” in the supporting information. The peak fractions of Mcm2-7 (0.6 pmol) and Mcm4/6/7 (1.5 pmol) were separated by 10% PAGE and stained with Coomassie brilliant blue R (Sigma-Aldrich).

Fig. 4.13: Influence of ATP levels on the hCMG helicase activity and its binding to oligonucleotide substrates. The helicase and DNA binding activities of the hCMG complex (20 fmol) were measured in the presence of different ATP levels. The amount of substrate unwound (%) and bound (%) were determined and plotted against the level of ATP added. The substrate was prepared by annealing oligonucleotides #16 (labeled) with #17.

20 Supplementary Figure 1-7 Influence of ATP levels on the hCMG helicase activity and its binding to oligonucleotide substrates. The helicase and DNA binding activities of the hCMG complex (20 fmol) were measured in the presence of different ATP levels. The amount of substrate unwound (%) and bound (%) were determined and plotted against the level of ATP added. The substrate was prepared by annealing oligonucleotides #16 (labeled) with #17.

Supplementary Figure 1-8 Purification of Mcm2-7 and Mcm4/6/7 complex. The Mcm2-7 and Mcm4/6/7 complexes were purified as described in “Materials and Methods” in the supporting information. The peak fractions of Mcm2-7 (0.6 pmol) and Mcm4/6/7 (1.5 pmol) were separated by 10% PAGE and stained with Coomassie brilliant blue R (Sigma-Aldrich).

Fig. 4.14: Purification of Mcm2-7 and Mcm4/6/7 complex. The Mcm2-7 and Mcm4/6/7 complexes were purified as described in ”Materials and Methods” in the supporting information. The peak fractions of Mcm2-7 (0.6 pmol) and Mcm4/6/7 (1.5 pmol) were separated by 10% PAGE and stained with Coomassie brilliant blue R (Sigma-Aldrich).

Chapter 5 Discussion

Faithful DNA replication is crucial for successful genome preservation. The components for replication are essential for cell division, and are difficult to examine. As a result, many replication proteins on leading and lagging strand are yet to be characterized. Lagging strand replication is initiated by DNA primase that synthesizes primers. In chap. 3 we describe the biochemical properties of the DNA primase isolated from the euryarchaeon, Thermococcus kodakaraensis. The complex utilizes both dNTPs and rNTPs to start chains (fig. 3.2). The catalytic subunit p41 initiates primers in the presence of dNTPs, but is nearly inactive with rNTPs. The initiation of chains with dNTPs differs from those formed with the eukaryotic or prokaryotic primases, which start chains only with rNTPs ([185] and fig. 3.8). However, it is in accord with a report using the primase complex of Pyrococcus furiosus (Pf) [116]. In keeping with observations made with the Pf p41/p46 complex, the addition of dATP inhibits RNA synthesis, while DNA synthesis is stimulated slightly by rATP addition (fig. 3.5). These findings suggest that the Tk primase complex preferentially utilizes dNTPs. The Km values for rNTPs and dNTPs verified that the affinity of the tk primase for dNTPs is slightly higher than that for rNTPs. Similar to that reported for the primase complex of the euryarchaeal Pf p41/p46 complex [115, 116].

In contrast, the primase complex isolated from the crenarchaeon Sulfolobus solfataricus was reported to possess a substantially greater affinity for rNTPs than dNTPs [190]. The question as to which nucleotides start chains in vivo remains to be answered.

In a comparison of the complex with the catalytic subunit alone, the complex forms shorter chains, ranging up to about 1 kilobase, while the p41 subunit products range up to 6 kilobases (fig. 3.1). The reduction of product length indicates a regulatory role of the larger subunit, p46, as described previously for the primase isolated fromMethanococcus

Im Dokument Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate (Seite 95-0)