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Nucleic Acids Research, 1988, Vol. 16, No. 13 5895-5914
© 1988

Articles

Characterization of the origins of replication of bacteriophage ø29 DNA

Julio Gutiérrez, Cristina Garmendia and Margarita Salas

Centra de Biología Molecular (CSIC-UAM), Universidad Autónoma Canto Blanco, 28049 Madrid, Spain

Received March 23, 1988. Revised June 10, 1988. Accepted June 10, 1988.

The origins of replication of ø29 DNA have been studied by analyzing the activity as templates in the ø29 in vitro replication system of E. coli recombinant plasmids and M13 derivatives containing ø29 DNA terminal sequences. Plasmid pITR, containing the 6 bp long inverted terminal repeat of ø29 DNA, was shown to be essentially inactive. The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right ø29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the terminal 12 bp at each DNA end. Point site-directed and random mutagenesis at these sequences was carried out. Changes of the second or third A into a C completely abolished the template activity. In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat. The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.


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