Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (αLIR and βLIR), which are essential elements for the two distally located R6K α and β DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5′ end of αLIR, whereas a gene coding for DDP3 is located at the 3′ end of βLIR. DDP1 and DDP2 are required for primary DNA distortion within αLIR or βLIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within αLIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.