Template specificity of Tk primase complex

2.8 Supplemental Data

3.4.4 Template specificity of Tk primase complex

We examined the template activity of the four homo-oligodeoxynucleotides, 30 nt in length, using the Tk primase complex. As shown in fig. 3.3 , oligo dC30 was the most effective template (compare panel A to panels B-D), supporting extensive DNA synthesis with dGTP (lanes 5 and 7). Under the conditions used, dGTP incorporation exceeded the level of oligo dC30 template added and the length of DNA chains, determined by urea-PAGE analyses, were >100 nt. Extensive RNA synthesis of a more homogeneous size (∼50 nt) than DNA was observed (fig. 3.3 (A), lanes 6 and 8). (It should be noted that the autoradiogram presented in fig. 3.3 (A) was exposed for 25 min at−80^◦C while all other autoradiograms shown in fig. 3.3 were exposed for 1 h at −80^◦C). Like oligo dC30, oligo dT30 supported extensive DNA and RNA synthesis (fig. 3.3 (B)). However, oligo dT30 supported the synthesis of shorter RNA chains than those formed in the pres-ence of oligo dC30 (fig. 3.3 (B), lanes 7 and 8) as well as the production of higher levels of RNA than DNA (fig. 3.3 (B), compare lanes 7 and 8 to lanes 3 and 4). In the presence of oligo dA30 (fig. 3.3 (C)) or oligo dG30 (fig. 3.3 (D)) DNA synthesis was detected with the former but barely discernable with the latter while RNA synthesis with these purine oligonucleotide templates was not observed. Thus, like eukaryotic DNA primases, the Tk primase complex prefers pyrimidine templates [185]. DNA products formed in reactions containing oligo dC30 or oligo dT30 and the p41 subunit in lieu of the complex, were sim-ilar to those shown in fig. 3.3 (A) and fig. 3.3 (B), respectively. The level of incorporation with the catalytic subunit alone was 20-30% lower than that detected with the complex.

3.4. Results 45

10 Figure 1.2 Analysis of DNA and RNA products formed by the Tk primase complex. Reactions (100 µl), as described in Fig. 1A containing 100 µM each of GTP, CTP, UTP and [!³²P]-ATP or 100 µM each of dGTP, dCTP, dTTP and [!³²P]-dATP, 417 fmol of ssM13 and 56 pmol of Tk primase complex were incubated for 20 min at 60°C.

Approximately 700 pmol of [!³²P]-dATP and 500 pmol of [!³²P]-ATP were incorporated. Mixtures were adjusted to 0.02 M EDTA, 1% SDS and following addition of 10 µg proteinase K incubated for 30 min at 37°C and then extracted consecutively with 100 µl of phenol-CHCI3-isoamyl alcohol mixture (25:24:1, v/v) and 100 µl of CHCl3 – isoamyl alcohol (24:1 v/v). Aqueous phases were adjusted to 1 M ammonium acetate and treated with 2 volumes of isopropanol. After 30 min on dry ice, the mixtures were centrifuged for 30 min at 20,000 rpm in the Eppendorf centrifuge at 4°C and pelleted material washed with 50% isopropanol, dried in vacuo and suspended in 25 µl TE buffer containing 50 mM NaCl. Reactions (10 µl) containing 2 µl of the RNA or DNA products, isolated as described above, were incubated with pancreatic DNase (0.1 µg) or pancreatic RNase (1 µg) in mixtures containing 40 mM Tris-HCl (pH 7.5) and 1 mM magnesium acetate; reactions with nuclease P1 (0.3 unit) contained 40 mM sodium acetate buffer (pH 5.2); reaction with CIP (1 unit) contained 40 mM Tris-HCl (pH 8.8) and 1 mM magnesium acetate. All reactions were incubated for 30 min at 37°C. Controls included incubation of products at 37°C for 30 min at the indicated pH in the absence of enzymes. Aliquots of reactions were subjected to urea-PAGE separation and analysis as described in Fig. 1. A. Digestion of DNA product. B. Digestion of RNA products. The isolated RNA products were incubated on ice (lane 1) or at 37ºC in the absence of enzymes (lane 2). Incubation with the indicated enzymes were carried out as described above. Treatment of RNA products with CIP was omitted.

Fig. 3.2: Analysis of DNA and RNA products formed by the Tk primase complex.

Reactions (100µl), as described in fig. 3.1 (A) containing 100µM each of GTP, CTP, UTP and [α-³²P]-ATP or 100µM each of dGTP, dCTP, dTTP and [α-³²P]-dATP, 417 fmol of ssM13 and 56 pmol of Tk primase complex were incubated for 20 min at 60^◦C. Approximately 700 pmol of

[α-32P]-dATP and 500 pmol of [α-³²P]-ATP were incorporated. Mixtures were adjusted to 0.02 M EDTA, 1% SDS and following addition of 10µg proteinase K incubated for 30 min at 37^◦C and then extracted consecutively with 100µl of phenol-CHCI3-isoamyl alcohol mixture (25:24:1, v/v) and 100µl of CHCl3 ˆa ˘A¸S isoamyl alcohol (24:1 v/v). Aqueous phases were adjusted to 1 M ammonium acetate and treated with 2 volumes of isopropanol. After 30 min on dry ice, the mixtures were centrifuged for 30 min at 20,000 rpm in the Eppendorf centrifuge at 4^◦C and pelleted material washed with 50% isopropanol, dried in vacuo and suspended in 25µl TE buffer containing 50 mM NaCl. Reactions (10µl) containing 2µl of the RNA or DNA products, isolated as described above, were incubated with pancreatic DNase (0.1µg) or pancreatic RNase (1µg) in mixtures containing 40 mM Tris- HCl (pH 7.5) and 1 mM magnesium acetate; reactions with nuclease P1 (0.3 unit) contained 40 mM sodium acetate buffer (pH 5.2); reaction with CIP (1 unit) contained 40 mM Tris-HCl (pH 8.8) and 1 mM magnesium acetate. All reactions were incubated for 30 min at 37^◦C. Controls included incubation of products at 37^◦C for 30 min at the indicated pH in the absence of enzymes. Aliquots of reactions were subjected to urea-PAGE separation and analysis as described in fig. 3.1. (A) Digestion of DNA product. (B) Digestion of RNA products.The isolated RNA products were incubated on ice (lane 1) or at 37^◦C in the absence of enzymes (lane 2). Incubation with the indicated enzymes were carried out as described above. Treatment of RNA products with CIP was omitted.

When M13 DNA was used in lieu of the short homopolymers (in reactions containing all four rNTPs or the four dNTPs), all labeled rNTPs and dNTPs supported RNA and DNA synthesis, respectively (data not presented). As shown in fig. 3.3, DNA products formed

46 3. Characterization of the DNA primase complex

12 and partial re-annealing reactions contributed to the synthesis of DNA chains longer than the DNA template added.

Figure 1.3 A-D. Template specificity of Tk primase complex. Reaction mixtures were as described in Materials and Methods and included. 100 µM of indicated [!³²P]-dNTP or [!³²P]-rNTP, 10 or 30 pmol of oligo dC30 (A); oligo dT30 (B); oligo A30 (C); oligo dG30 (D), and 5.6 pmol of the Tk primase complex. Following incubation for 20 min at 60°C, aliquots were subjected to urea-PAGE separation. The specific activity (cpm/pmol) of the [!32P]-labeled nucleotides used were: dTTP, 2580; UTP, 2020; dGTP, 2280; GTP, 1640; dCTP, 2285; CTP, 1900; dATP 1840; ATP, 1710.

12 and partial re-annealing reactions contributed to the synthesis of DNA chains longer than the DNA template added.

Fig. 3.3: A-D. Template specificity of Tk primase complex. Reaction mixtures were as described in Materials and Methods and included. 100µM of indicated [α-³²P]-dNTP or

[α-32P]-rNTP, 10 or 30 pmol of oligo dC30 (A); oligo dT30 (B); oligo A30 (C); oligo dG30 (D), and 5.6 pmol of the Tk primase complex. Following incubation for 20 min at 60^◦C, aliquots were subjected to urea-PAGE separation. The specific activity (cpm/pmol) of the [α-³²P]-labeled nucleotides used were: dTTP, 2580; UTP, 2020; dGTP, 2280; GTP, 1640; dCTP, 2285; CTP, 1900; dATP 1840; ATP, 1710.

with some of the 30 nt oligonucleotide templates were longer in length than the template added. This discrepancy could arise by mechanisms that minimally include slippage, displacement synthesis and primer-template re-annealing followed by chain extension or terminal addition of nucleotides to template ends. The latter mechanism was examined with oligo dA30. Incubation of the primase complex with [α-³²P]-dideoxy TTP (ddTTP) and oligo dA30 did not result in the production of labeled DNA chains. Supplementation of reactions with terminal deoxynucleotidal transferase led to the addition of the labeled ddTTP (data not presented). These findings suggest that the Tk primase complex did not support terminal addition under the conditions used. All products formed (with M13 or homopolymers) were hydrolyzed by nuclease P1 (>90%), a single strand specific nuclease (data not presented), suggesting that extensive duplex DNA or duplex DNA-RNA hybrid structures were not formed under the conditions used. Since Tk primase reactions were carried out at 60^◦C, we speculate that melting and partial re-annealing reactions contributed to the synthesis of DNA chains longer than the DNA template added.

3.4. Results 47

3.4.5 Examination of the 5’ ends of RNA and DNA chains formed

Im Dokument Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate (Seite 62-65)