P32 αdctp

The Pol-prim assay measures primase-initiated DNA synthesis on unprimed Φ174 ssDNA as previously described using SV40 DNA replication conditions (36 (link),57 (link)) with slight modifications. In short, 40 μl of assay Tag samples (full length protein or shortened monomer variants as indicated) were incubated in buffer containing 30 mM HEPES–KOH, pH 7.8, 7 mM MgAc, 0.1 mM EGTA, 1 mM DTT, 0.25 mg/ml BSA, 0.01 mg/ml creatine kinase, 40 mM creatine phosphate, 4 mM ATP, 0.2 mM each of CTP, GTP and UTP, 100 μM of each dATP, dGTP and dTTP plus 50 μM dCTP in the presence of 0.1 μCi P32 α-dCTP (3000 Ci/mmol, Perkin Elmer) and 250 ng Φ174-ssDNA template (0.76 nmol nucleotides, NEB). Incubations were carried out in the absence or presence of RPA (0.5 μg, unless otherwise stated). The amounts of full length Tag and variant proteins in the reactions are indicated individually. The reaction mixture was assembled on ice, and the reaction was started by the addition of 10 ng of human Pol-prim. After incubation for 1 h at 37°C, the reaction was spotted on Whatman GF-C glass fiber filters and submerged in 10% trichloroacetic acid as previously described to measure the amount of incorporated nucleotides (57 (link),58 (link)).

Independent Lys⁺ colonies were patched on SD-LYS plates, and their DNA was extracted from a saturating overnight 5 mL SD-LYS liquid culture. DNA was digested by HindIII (for the inter-chromosomal donor construct), PstI (for the ectopic S2-70bp donor construct), or PstI+EcoRI (for the ectopic S2-1000bp donor construct) 4 hours at 37° C and migrated overnight in 0.8% Agarose-LE (Affymetrix) in 1X TBE at 50 V. The DNA was transferred from the gel onto an Amersham Hybond-XL membrane (GE healthcare) following the manufacturer instructions (alkali protocol). The membrane was blocked with Church buffer (1% BSA, 0.25 M Na₂HPO₄ pH7.3, 7% SDS, 1 mM EDTA) for 2–3 hrs at 65° C. The LY, S2, or LYS2 probes (2, 2, and 4 kb-long, respectively) together with Phage λ DNA (molecular ladder) were radio-labeled by random priming with P³²-αdCTP (6,000 Ci/mmol; Perkin-Elmer) using the Decaprime II kit (Ambion Inc) and incubated with the membrane overnight at 65° C. After 3 – 5 washes for 10 min at 65° C (20 mM Na₂HPO₄ pH 7.3, 1% SDS, 1 mM EDTA), membranes were exposed for 8 to 24 hours, and the Storage Phosphor Screen (GE healthcare) scanned on a Storm Phosphorimager (Molecular Dynamics).