Table 1.
Dissociation constant (Kd) of HPF1 mutants binding to PARP1 CAT ΔHD and their effects on ADP-ribosylation of full-length PARP1 (automodification), histone H3, and HPF1.
| Location of HPF1 mutants | Kd(μM)a | ADP-ribosylationb | ||
|---|---|---|---|---|
| PARP1 | H3 | HPF1 | ||
| Control | ||||
| Wild-type |
1.5 ± 0.2 2.8 ± 0.3c |
− | ++ | − |
| Interface Id | ||||
| F268S | N.D. | + + + | ++ | − |
| F280A | 6.0 ± 0.8 | +++ | ++ | − |
| D283H | N.D. | + + + | + | − |
| C285H | 28.7 ± 1.6 | +++ | ++ | − |
| K307S | 9.3 ± 1.2 | ++ | ++ | − |
| Interface IId | ||||
| E138K | 1.7 ± 0.1 | − | ++ | − |
| F139S | 2.4 ± 0.1 | − | ++ | − |
| K216E | 1.4 ± 0.1 | − | ++ | − |
| Active center | ||||
| R239A | 2.4 ± 0.1 | +++ | + | +++ |
| E284A | 0.3 ± 0.0 | + | − | + |
aKd data were determined in isothermal titration calorimetric (ITC) assays. The measurements were conducted in duplicates and the standard error values were listed. See Supplementary Fig. 2 and 3 for raw data.
bThe assessment was made based on the ADP-ribosylation assays (see Fig. 2b). Number of “+” indicates the relative intensity of observed ADP-ribosylation.
cTitration of HPF1 to full-length PARP1 in the presence of DNA. Other Kd values listed in this table are for titration of HPF1 to PARP1-CAT ΔHD.
dThese interfaces were observed in the HPF1/PARP1-CAT ΔHD complex structure, indicating the two candidate interaction modes between HPF1 and PARP1.