TABLE 2.
Primase-coupled polymerase activity with the herpes polymerase (UL30 or UL30-UL42)
Primase-coupled polymerase activity (% primers utilized and % primers transferred) was measured for each primase/polymerase pair at equivalent molar concentrations of each enzyme with the exception of studies using UL5-UL52 that contained 400 nm UL5-UL52 and 100 nm polymerase.
| Primase | Polymerase | DNA template | Primers utilized | Primers transferred intramolecularlya |
|---|---|---|---|---|
| % | % | |||
| UL5-UL52-UL8 | UL30-UL42 | T20GTCCT36 | 0.6 ± 1.1 | 0.039 ± 0.026 |
| UL5-UL52-UL8 | UL30 | T20GTCCT36 | 0.3 ± 0.1 | NDb |
| UL5-UL52-UL8 (Hel-) | UL30-UL42 | T20GTCCT36 | 1.6 | 0.26 |
| UL5-UL52-UL8 (Hel-) | UL30 | T20GTCCT36 | 0.3 | ND |
| UL5-UL52 | UL30-UL42 | T20GTCCT36 | <0.2 | ND |
| UL5-UL52 + UL8 | UL30-UL42 | T20GTCCT36 | 0.9 ± 0.5 | 0.036 |
| UL5-UL52c | UL30-UL42 | T20GTCCT36 | 0.0015 ± 0.0005 | ND |
| UL5-UL52 + UL8c | UL30-UL42 | T20GTCCT36 | 0.34 ± 0.07 | 0.025 ± 0.018 |
| UL5-UL52/UL8 | UL30-UL42 | T20GCCCCAT17 | 3.6 ± 0.4 | 0.94 |
| UL5-UL52-UL8 (Hel-) | UL30-UL42 | T20GCCCCAT17 | 2.6 | 0.23 |
| UL5-UL52c | UL30-UL42 | T20GCCCCAT17 | 0.008 ± 0.002 | 0.004 ± 0.0005 |
| UL5-UL52 + UL8c | UL30-UL42 | T20GCCCCAT17 | 0.34 ± 0.02 | 0.05 ± 0.01 |
| UL5-UL52-UL8 | UL30-UL42 | C20GTCCA19 | <0.2 | ND |
| UL5-UL52-UL8 | UL30-UL42 | (TCTG)15 | 0.4 | ND |
The percentage of primers transferred was calculated as a percentage of the primers utilized by the polymerase in each case, i.e. of the 0.6% of the primers synthesized by UL5-UL52-UL8 and further elongated by UL30/UL42 on 3′-d(T20GTCCT36)-5′, only 0.039% were transferred intramolecularly to UL30-UL42.
ND indicates no detectable products in the presence of binase.
These assays contained 1 μm dNTPs instead of 10 μm dNTPs to increase the specific activity of the [α-32P]dNTPs in order to allow quantitation of primase-coupled polymerase activity.