Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage lambda O replication initiator

Abstract

The bacteriophage lambda O protein binds to the lambda replication origin (orilambda) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to orilambda is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production of crystals of the origin-binding domain of lambda O that diffract to 2.5 A is reported. Anomalous dispersion methods will be used to solve this structure.

Figure 1

(a) Analysis of the purification of the ‘native’ λ OBD by SDS–PAGE. The protein samples were electrophoresed through a 15% polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane 1, uninduced cells; lane 2, induced cells; lane 3, fraction I; lane 4, fraction II; lane 5, fraction II (post-dialysis); lane 6, fraction III. (b) Gel mobility-shift assay of binding of the ‘native’ λ OBD to an oligonucleotide containing a λ O recognition sequence (see text). Increasing amounts of the λ OBD were incubated with a ³²P-labeled oligonucleotide at 303 K and the mixture was then subjected to electrophoresis in a native 8% polyacrylamide gel. The molar ratios of protein, as dimer, to DNA were lane 1, 0 (i.e. no protein added); lane 2, 0.2; lane 3, 0.5; lane 4, 1; lane 5, 5.

Figure 2

Crystals of ‘native’ and selenomethionine forms of the λ OBD. (a) Crystals of ‘native’ λ OBD. (b) Mounting a crystal of the selenomethionine-containing λ OBD using a 0.3 mm loop. Crystals measure 70 × 150 µm and are 20 µm thick.

Figure 3

Diffraction image obtained from a crystal of selenomethionine-containing λ OBD. These crystals diffract to a 2.5 Å resolution limit.