Table 2 Binding affinities of HaloTag-635-LIG3α-XRCC1-YFP with 601-NCP substrates
From: The zinc finger of DNA ligase 3α binds to nucleosomes via an arginine anchor
PROTEIN | DNA (601-NCP) | LIFETIMES (s) | koff (s–1) | N | kon app (s–1) | N’ | kon (M–1s–1) | KD (nM) |
|---|---|---|---|---|---|---|---|---|
Halo-LIG3α | UD-NCP | 24 ± 0.6 | 0.05 ± 0.001 | 99 | 0.1 ± 0.007 | 66 | 1.3 ± 0.08 ×109 | 0.04 ± 0.002 |
YFP-XRCC1 | UD-NCP | 34 ± 0.04 | 0.04 ± 0.001 | 100 | 0.08 ± 0.006 | 78 | 6.7 ± 0.5 ×107 | 0.60 ± 0.05 |
Halo-LIG3α | NO NCP 3’OH 5’OH | 2.0 ± 0.1 | 0.5 ± 0.02 | 77 | 0.03 ± 0.0008 | 68 | 1.9 ± 0.04 ×108 | 2.5 ± 0.1 |
YFP-XRCC1 | NO-NCP 3’OH 5’OH | 2.9 ± 0.6 | 0.4 ± 0.02 | 38 | 0.02 ± 0.001 | 29 | 3.6 ± 0.4 ×107 | 9.9 ± 1.1 |
Halo-LIG3α | SHL –4.5 | 17 ± 3 τfast = 2.3 (66%) τslow = 43.6 (34%) | 0.06 ± 0.001 | 115 | 0.07 ± 0.003 | 51 | 4.5 ± 0.1 ×108 | 0.13 ± 0.006 |
YFP-XRCC1 | SHL –4.5 | 11 ± 2 τfast = 2.0 (66%) τslow = 23.2 (34%) | 0.1 ± 0.02 | 171 | 0.08 ± 0.003 | 64 | 1.3 ± 0.05 ×108 | 0.75 ± 0.15 |
Halo-LIG3α | SHL −2.5 | 12 ± 4 τfast = 2.1 (76%) τslow = 42.0 (24%) | 0.09 ± 0.007 | 149 | 0.04 ± 0.001 | 54 | 2.3 ± 0.07 ×108 | 0.38 ± 0.03 |
YFP-XRCC1 | SHL -2.5 | 14 ± 0.8 τfast = 2.5 (52%) τslow = 21.5 (48%) | 0.09 ± 0.006 | 236 | 0.1 ± 0.007 | 59 | 2.4 ± 0.2 ×107 | 0.36 ± 0.03 |
Halo-LIG3α | SHL 0 | 10 ± 2 τfast = 2.6 (62%) τslow = 21.8 (38%) | 0.1 ± 0.02 | 91 | 0.03 ± 0.003 | 25 | 3.4 ± 0.3 ×108 | 0.28 ± 0.06 |
YFP-XRCC1 | SHL 0 | 13 ± 6 τfast = 4.5 (73%) τslow = 27.5 (27%) | 0.09 ± 0.06 | 83 | 0.2 ± 0.03 | 34 | 1.8 ± 0.3 ×108 | 0.53 ± 0.3 |
Halo-ZnF-BRCT LIG3α | UD-NCP | 11 ± 4 τfast = 3.7 (69%) τslow = 26.5 (31%) | 0.09 ± 0.03 | 107 | 0.08 ± 0.003 | 47 | 8.1 ± 0.3 ×107 | 1.1 ± 0.3 |
YFP-XRCC1 | UD-NCP | 16 ± 4 τfast = 3.3 (65%) τslow = 30.4 (35%) | 0.08 ± 0.02 | 77 | 0.4 ± 0.3 | 52 | 4.9 ± 3 x 108 | 0.16 ± 0.1 |
Halo-Del-ZnF LIG3α | UD-NCP | NO | NO | NO | NO | NO | NO | NO |
YFP-XRCC1 | UD-NCP | NO | NO | NO | NO | NO | NO | NO |
Halo-R62A-R64A LIG3α | UD-NCP | NO | NO | NO | NO | NO | NO | NO |
YFP-XRCC1 | UD-NCP | NO | NO | NO | NO | NO | NO | NO |
Halo-K421A- LIG3α | SHL-2.5 | 2.5 | 0.40 | 18 | NO | NO | NO | NO |
YFP-XRCC1 | SHL–2.5 | 2.0 | 0.50 | 11 | NO | NO | NO | NO |
