EcoRII: a restriction enzyme evolving recombination functions?

Abstract

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5'CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two-domain structure that enables this particular mode of protein-DNA interaction. The C-terminal domain is a new restriction endonuclease, EcoRII-C. In contrast to the wild-type enzyme, EcoRII-C cleaves DNA specifically at single 5'CCWGG sites. Moreover, substrates containing two or more cooperative 5'CCWGG sites are cleaved much more efficiently by EcoRII-C than by EcoRII. The N-terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5'CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA-binding domain to enable the interaction with two 5'CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons.

Fig. 1. (A) Digestion of EcoRII by trypsin in the presence or absence of specific DNA. A, B and C are proteolytic fragments: A, 33–34 kDa; B, 23 kDa; and C, ∼30 kDa. Digestion times are given at the top of each lane. M, pre-stained protein molecular weight marker (New England Biolabs). Molecular weights were estimated using the Broad Range molecular weight marker (New England Biolabs) not shown here. (B) Digestion of EcoRII by chymotrypsin in the presence or absence of specific DNA. D and E are proteolytic fragments: D, 26–27 kDa; and E, 21–22 kDa. Digestion times are given at the top of each lane. M, Broad Range protein molecular weight marker (New England Biolabs).

Fig. 2. Top: identification of the proteolytic cleavage fragments by Edman degradation. Possible cleavage positions in the EcoRII primary sequence were determined from the molecular weight of the fragments in SDS–polyacrylamide gels. Bottom: assignment of the proteolytic fragments to the EcoRII sequence. The amino acid sequences of the N-terminal fragments B and E start with the His₆ tag of the protein. The length of the bars represents the length of the EcoRII sequence (404 amino acids) and of the proteolytic fragments, respectively.

Fig. 3. Electrophoretic mobility shift assay with wild-type EcoRII and EcoRII-N in the presence of a 191 bp DNA molecule (0.6 nM) containing a single 5′CCWGG site. Enzyme concentrations c (nM) are indicated at the top of each lane.

Fig. 4. (A) Kinetics of the cleavage reactions of wild-type EcoRII and EcoRII-C with linearized pBR322 Dcm^– DNA. The reaction times are given at the top of each lane. BstNI, an isoschizomer of EcoRII (positive control); M, molecular weight marker. (B) Cleavage of T3 DNA with EcoRII-C and wild-type EcoRII. Enzyme amounts are given at the top of each lane. Left lane, T3 DNA without enzyme; BstNI, positive control. Molecular weight markers are given on the right.

Fig. 5. Fraction (%) of the dimeric form of EcoRII-N and EcoRII-C determined by analytical ultracentrifugation. Open circles, EcoRII-N; filled circles, EcoRII-C; graph without data points, wild-type EcoRII for comparison as determined previously (Behlke et al., 1997).