Table 2. Requirements for ATPase activity of hFBH1.
| Additions or omissions | Amount added | Relative activity (%)a |
|---|---|---|
| Completeb |
|
100 |
| Add human RPA |
0.2, 0.4, 0.6 µg |
105, 104, 98 |
| Add E.coli ssb |
0.2, 0.4, 0.6 µg |
67, 38, 47 |
| Add NaCl |
0.05, 0.1, 0.2 M |
122, 112, 107 |
| Omit MgCl2 |
|
<1 |
| Omit DTT + NEMc |
10 mM |
<1 |
| Omit ATP |
|
|
| Add GTP or CTP or UTP |
250 µM each |
15, 40, 52 |
| Add dATP |
250 µM |
110 |
| Add dGTP or dCTP or dTTP |
250 µM each |
83, 49, 5 |
| Omit φX174 sscDNA |
|
9 |
| Add φX174 sscDNA |
1, 5, 20 ng |
36, 98, 102 |
| Add poly(dI–dC) double strand |
50 ng |
89 |
| Add tRNA |
50 ng |
16 |
| Add φX174 RFI |
50 ng |
67 |
| Add φX174 RFIII | 50 ng | 34 |
aThe value of 100% in this experiment corresponded to the hydrolysis of 1620 pmol ATP.
bThe complete reaction contained 75 fmol of GST–hFBH1 in the standard reaction mixture described in Materials and Methods. ATP hydrolysis was measured as described in Materials and Methods.
cThe enzyme was first incubated for 10 min at 37°C in the presence of the indicated amount of NEM.