TABLE 3.

Mutations that disrupt the dimer interface result in a loss of processivity

Dimer interface disrupting mutants have a decrease in activity (k_cat), an increase in K_m for DNA and AdoMet, a large increase in the rate of DNA dissociation, and a loss of processivity. k_cat and K_m were determined by monitoring the ability of the enzyme to incorporate tritiated methyl groups transferred from cofactor AdoMet onto DNA (poly-dIdC). k_off values were determined by binding excess enzyme to 5′ FAM-6 labeled GCbox30 duplex and measuring the rate of change in anisotropy upon the addition of excess unlabeled GCbox30 (100-fold labeled DNA). Processivity n½ was determined by mathematical modeling of the activity curve.

Substrate	poly-dIdC		RASSF1A		poly-dIdC		poly-dIdC		GCbox30		poly-dIdC		RASSF1A
	kcat	Fold ↓	kcat	Fold ↓	KmAdoMet	Fold ↑	KmDNA	Fold ↑	koff	Fold ↑	Processivity	Fold ↓	Processivity	Fold ↓
	h−1		h−1		μm		μm		min−1
WT	3.5 ± 0.27	1.0	1.39 ± 0.06	1.0	0.20 ± 0.02	1.0	1.2 ± 0.1	1.0	0.21 ± 0.01	1.0	30 ± 4.5	1.0	15 ± 5.0	1.0
H873A	1.02 ± 0.16	3.4	0.60 ± 0.16	2.3	0.90 ± 0.07	4.4	4.9 ± 0.7	3.9	3.48 ± 0.29	16	2.4 ± 1.5	13	1.7 ± 0.5	9.1
H873R	0.26 ± 0.08	13	0.22 ± 0.09	6.3	1.17 ± 0.18	5.8	5.0 ± 0.8	4.0	4.17 ± 0.78	20	1.4 ± 0.7	21	0.9 ± 0.4	17
W860A	0.34 ± 0.08	10	0.31 ± 0.05	4.4	1.41 ± 0.28	6.9	4.8 ± 0.6	3.9	3.07 ± 0.19	14	2.0 ± 1.1	15	0.9 ± 0.3	17
N879A	0.73 ± 0.15	4.7	0.71 ± 0.04	1.9	0.81 ± 0.06	4.0	3.7 ± 0.7	3.0	1.52 ± 0.13	7.2	2.4 ± 1.7	12	2.1 ± 0.5	7.3
R882H	1.41 ± 0.15	2.5	0.75 ± 0.09	1.8	1.30 ± 0.14	6.4	6.6 ± 0.6	5.3	2.36 ± 0.04	11	2.2 ± 1.9	14	2.1 ± 0.7	7.3
D876G	1.43 ± 0.46	2.4	0.78 ± 0.14	1.8	0.57 ± 0.02	2.8	3.4 ± 0.3	2.7	1.35 ± 0.10	6.4	3.8 ± 2.2	7.9	1.9 ± 0.3	8.2
R885A	No activity		No activity		NA		NA		11.2 ± 2.32	53	NA		NA