Supplemental data - Working towards understanding DNA replication : coupling of a 3'-5' helicas

24 Table 1.1 Coupling of Tk DNA Primase activity with Klenow Pol. Reaction mixtures (20ml), containing 20 mM, Tris-HCl (pH 8.0), 2.5 mM DTT, 3 mM magnesium acetate, 100 mg/ml BSA, 50 mM [!³²P]-dATP (2070 cpm/pmol), 0.4 unit of Klenow, 4.54 pmol of poly dT₂₂₁ and 50 mM NaCl were incubated for 30 min at 37ºC. Reactions with the human primase complex (p58 + p48 subunits) also included 2.5 mM ATP, where indicated. Aliquots of each reaction were plated on DEAE cellulose paper and washed three times with 0.5 M ammonium formate + 1 mM sodium pyrophosphate, dried and counted.

Additions Protein added dAMP incorporated

(fmol) (pmol)

I. Wild-type complex (p41/p46) 40 4 0.4

684 72.4 10.4 II. Mutant complex (m p41/p46) 40

VIII. human primase complex +ATP - ATP

70 70

716

Fig. 3.8: Coupling of Tk DNA Primase activity with Klenow Pol. Reaction mix-tures (20 ml), containing 20 mM, Tris-HCl (pH 8.0), 2.5 mM DTT, 3 mM magnesium acetate, 100 mg/ml BSA, 50 mM [α-³²P]-dATP (2070 cpm/pmol), 0.4 unit of Klenow, 4.54 pmol of poly dT221 and 50 mM NaCl were incubated for 30 min at 37^◦C. Reactions with the human primase complex (p58 + p48 subunits) also included 2.5 mM ATP, where indicated. Aliquots of each reaction were plated on DEAE cellulose paper and washed three times with 0.5 M ammonium formate +1 mM sodium pyrophosphate, dried and counted.

3.7 Supplemental data

58 3. Characterization of the DNA primase complex

25 1.7 Supplemental data

Supplementary Figure 1-1 Oligo dA chains contain 5-phosphate ends Oligo dA products were prepared as described in Fig. 4B from reactions that were increased 10-fold. Following incubation, the mixture was supplemented with 100 µg of BSA, 100 µl H20 and treated with 100 µl of 7% HClO4. After 20 min on ice, the mixture was centrifuged for 30 min at 4ºC in an Eppendorf centrifuge. The pellet was dissolved in 100 µl of 0.1 N NaOH and then precipitated with 100 µl of 7% HClO⁴. The pellet was collected and the procedure repeated twice; the final pellet was dissolved in 100 µl of 0.05 N NaOH. The ³²P present in oligo dA (determined by DEAE paper) pre- and post-acid precipitation were identical. Degradation of the DNA product was carried out as follows: a reaction mixture (25 µl) containing 20 mM Tris-HCl (pH 8.0), 2 mM CaCl2, 10 µl of the oligo dA (1.5x10⁶ cpm, equivalent to ~1.5 nmol of ³²P-dAMP) and micrococcal nuclease (125U, Worthington) was incubated at 37ºC for 60 min. The mixture was then adjusted to pH

~7.0 with 0.93 µl of 1N HCl and treated with 0.5 U of spleen phosphodiesterase. Following 30 min at 37ºC, an additional 0.25 U of spleen phosphodiesterase was added and the mixture incubated for an additional 30 min. We verified that the hydrolysis of the oligo dA chains was complete (monitored by CIP treatment which indicated that more than 95% of the radioactivity was converted to ³²Pi). Aliquots from the nuclease digested material were then incubated with and without CIP as follows: reactions mixtures (5µl) containing 2.5 µl of the digested material, 1 mM magnesium acetate, 20 mM Tris-HCl (pH 8.0) were incubated with and without CIP (2U) for 30 min at 37ºC. Aliquots (1µl) of the treated material was subjected to TLC separation on separate PEI strips that were developed in 0.5 M ammonium formate (pH 3.5). Prior to loading, pAp (3') and dAp (3') (5 µmol each) were added to the reaction as markers. The markers were located on the plates and the distribution of 32P monitored by counting of 1 cm strips of the PEI plate. This analysis revealed ³²P peaks corresponding to the pdAp and dAp markers only in the digest not treated with CIP. Quantitation of the ³²P present in the dA and pdAp regions revealed the presence of 70,000 and 2200 cpm, respectively. The oligo dA chains formed by Tk DNA primase, determined by size and amount of [!³² P]-dATP incorporated, were calculated to be between 30 and 40 nt in length. Hence, based on the amount of ³²P detected in the monophosphate region and the length of the oligo dA chains, we calculated that the pdAp region (possessing two phosphate residues) should contain 4660 (30 nt chain) or 3500 cpm (40 nt chain) if we assume that all termini contained a 5' phosphate end. The observed recovery (2200 cpm) represents 47-63% of the calculated value (~50%).

2

Fig. 3.9: Oligo dA chains contain 5-phosphate ends (A) Model of the synthesis of leading and lagging strand synthesis. Oligo dA products were prepared as described in fig. 3.4 (B) from reactions that were increased 10-fold. Following incubation, the mixture was supplemented with 100µg of BSA, 100µl H₂0 and treated with 100µl of 7% HClO4. After 20 min on ice, the mixture was centrifuged for 30 min at 4^◦C in an Eppendorf centrifuge. The pellet was dissolved in 100µl of 0.1 N NaOH and then precipitated with 100µl of 7% HClO₄. The pellet was collected and the procedure repeated twice; the final pellet was dissolved in 100µl of 0.05 N NaOH. The ³²P present in oligo dA (determined by DEAE paper) pre- and post-acid precipitation were identical. Degradation of the DNA product was carried out as follows: a reaction mixture (25µl) containing 20 mM Tris-HCl (pH 8.0), 2 mM CaCl2, 10µl of the oligo dA (1.5x106 cpm, equivalent to ∼1.5 nmol of ³²P-dAMP) and micrococcal nuclease (125 U, Worthington) was incubated at 37^◦C for 60 min. The mixture was then adjusted to pH ∼7.0 with 0.93µl of 1 N HCl and treated with 0.5 U of spleen phosphodiesterase. Following 30 min at 37^◦C, an additional 0.25 U of spleen phosphodiesterase was added and the mixture incubated for an additional 30 min. We verified that the hydrolysis of the oligo dA chains was complete (monitored by CIP treatment which indicated that more than 95% of the radioactivity was converted to ³²Pi). Aliquots from the nuclease digested material were then incubated with and without CIP as follows: reactions mixtures (5µl) containing 2.5µl of the digested material, 1 mM magnesium acetate, 20 mM Tris-HCl (pH 8.0) were incubated with and without CIP (2U) for 30 min at 37^◦C. Aliquots (1µl) of the treated material was subjected to TLC separation on separate PEI strips that were developed in 0.5 M ammonium formate (pH 3.5). Prior to loading, pAp (3’) and dAp (3’) (5 ˆIijmol each) were added to the reaction as markers. The markers were located on the plates and the distribution of ³²P monitored by counting of 1 cm strips of the PEI plate. This analysis revealed ³²P peaks corresponding to the pdAp and dAp markers only in the digest not treated with CIP. Quantitation of the³²P present in the dA and pdAp regions revealed the presence of 70,000 and 2200 cpm, respectively. The oligo dA chains formed by Tk DNA primase, determined by size and amount of [α-³²P]-dATP incorporated, were calculated to be between 30 and 40 nt in length. Hence, based on the amount of ³²P detected in the monophosphate region and the length of the oligo dA chains, we calculated that the pdAp region (possessing two phosphate residues) should contain 4660 (30 nt chain) or 3500 cpm (40 nt chain) if we assume that all termini contained a 5’ phosphate end. The observed recovery (2200 cpm) represents 47-63% of the calculated value (∼50%).

3.7. Supplemental data 59

Supplemental Table 1.1 Sequence of oligonucleotides used to construct the 200-nt rolling circle substrate.

Supplemental Table 1.2 RNA and DNA synthesis with oligo dT30 as template Reaction mixtures (20 µl) containing 40 mM glycine (pH 8.9), 100 µM [!³²P]-ATP (665 cpm/pmol) or [!³²P]-dATP (2500 cpm/pmol), 1 µM oligo dT30, 10 mM magnesium acetate, 4 mM MnCl2, 100 µg/ml BSA, 1 mM DTT and Tk primase preparations diluted in TE+BSA (20 µg/ml) were incubated for 20 min at 60ºC. Aliquots were used to measure the level of polynucleotides synthesized.

Supplemental Table 1.3 Influence of CIP on oligo rA products Reaction mixtures (10 µl) containing 10 mM Tris-HCl (pH 8.5), 0.5 mM magnesium acetate, 2.83 pmol ["³²P]-ATP labeled oligo-rA product (17,400 cpm) or 10.8 pmol of [!³²P]-ATP labeled oligo rA (7860 cpm) were incubated with CIP (3U, where indicated) for 30 min at 37ºC. Aliquots were subjected to analysis using DE-81 paper.