Terminal region recognition factor 1, a DNA-binding protein recognizing the inverted terminal repeats of the pGKl...

The yeast linear DNA plasmids pGKl1 and pGKl2 contain inverted terminal repeats (ITRs) and terminal proteins covalently bound to the 5' termini of each plasmid. The presence of these features suggests a protein-primed mechanism of DNA replication, similar to that exemplified by mammalian adenovirus and phi 29 phage of Bacillus subtilis. In this paper, we report the identification of an activity in cytoplasmic extracts of yeast harboring the pGKl plasmids that recognizes the termini of both pGKl1 and pGKl2. We call this activity TRF1, for terminal region recognition factor 1. Deletion analyses and DNase I protection experiments demonstrate that the activity recognizes base pairs 107-183 within the ITR of pGKl1, and base pairs 126-179 within the ITR of pGKl2. The presence of T-tracts within these two regions, but otherwise dissimilar nucleotide sequences, suggests that TRF1 recognizes a common structural feature within the ITRs of the two plasmids. TRF1 has been partially purified from yeast cytoplasmic extracts and Southwestern analysis indicates that the apparent molecular mass of the protein is 16 kDa. By expressing three open reading frames from pGKl2 in Escherichia coli, we found that open reading frame 10 (ORF10) of pGKl2 encodes TRF1. The sequence of the ORF10 gene product indicates that TRF1 is a highly basic protein of small molecular mass. Comparison of TRF1 with other DNA-binding proteins known to recognize the terminal regions of linear DNAs, such as NFI and NFIII involved in adenovirus DNA replication, and phi 29 p6, involved in phi 29 DNA replication, indicates that TRF1 has a different mode of binding.