Table 1.

Quantitative analysis of in situ cleavage experiments

Mutant heterodimer preparation RSS substrate Metal % Conversionatranspreference (V/VI ratio)
I II III IV V VI
D600A Intact 12 (Fig. 4A)Mg 8.7 0.3 5.6 6.6 6.60.3 19
Intact 12 (expt 2) Mg 17 0.2 7.821 22 0.4 54
Prenicked 12 (Fig. 5A)Mn 39 0.6 18 32 28 0.7 38
Prenicked 12 (expt 2) Mn 38 0.5 16 4240 0.9 47
Prenicked 12 Mg 1.10.06 0.36 0.78 0.58 0.03 18
Intact 23b Mg 2.8 0.11.0 1.5 1.5 0.2 7
Prenicked 23b Mn 17 0.1 5.0 1.5 7.00.1 54
D708A Intact 12 (Fig. 4B) Mg 110.2 4.0 8.7 9.0 0.2 43
Intact 12 (expt 2) Mg 23 0.3 20 14 19 0.4 47
Prenicked 12 (Fig. 5B) Mn 59 1.0 3155 52 1.1 45
Prenicked 12 (expt 2) Mn44 0.4 13 41 42 0.6 73
Prenicked 12 Mg 0.73 0.00 0.31 0.710.65 0.03 21
Intact 23b Mg 12 0.2 6.4 6.66.1 0.2 27
Prenicked 23b Mn 35 0.7 11 24 23 0.2 104
E962A Intact 12 (Fig. 4C) Mg 16 0.14.0 14 15 0.2 78
Intact 12 (expt 2) Mg15 0.1 3.7 13 12 0.2 57
Intact 12 (expt 3)c Mg 16 5.7 9.4 1713 4.2 3
Prenicked 12 (Fig. 5C) Mn29 0.7 13 15 15 1.1 13
Prenicked 12 (expt 2) Mn 59 0.3 11 57 53 0.7 77
Intact 23b Mg 2.70.2 0.9 1.4 1.2 0.1 9
Prenicked 23b Mn 20 0.9 8.1 7.77.0 0.2 35
  • a Measured as percentage of predominant nicked (intact) or hairpin (prenicked) species present relative to total DNA in samples recovered from complexes containing RAG-1 heterodimers (I to VI) (Fig. 2).

  • b RAG-RSS complexes assembled in the presence of HMG-1 (see Materials and Methods).

  • c Nicked products found in samples II and VI likely arose from cross-contamination since they are found at similar levels in both samples and are not reproducibly observed in two other experiments. However, the possibility that they are attributed to Glu-962 independent activity, observed by others at elevated metal ion concentrations (20), cannot be formally excluded. In both cases, the actual trans preference may be much greater than threefold, either because the amount of cleaved product recovered overrepresents the actual cleavage activity in these samples (the former case) or because the cleavage product observed represents a basal activity present in every sample that can be subtracted from all the samples (the latter case).