Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.     

Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA

Initiation of DNA replication in plasmid R6K is potentiated by the plasmid-encoded 35 kd replication initiator protein. We had previously reported that the initiator bound to two regions of R6K DNA called Site I and Site II. Using DNAase I footprinting technique we have demonstrated that the initiator bound to seven tandem repeats of a 22 bp long sequence in Site I. In Site II, the initiator bound to a single repeat having the same consensus sequence and to two partial repeats that most likely overlap the promoter of the initiation protein cistron. Using dimethyl sulfate as a chemical probe, we have determined the purine residues of Site I and Site II that make contact with the initiator protein. The results show that eight out of nine contact points per repeat in Site I were located on one of the two strands of the DNA. The binding of the initiator to the Site II sequence could explain the observed autoregulation of the synthesis of the initiator protein by promoter occlusion.     

DNA sequence of the 5'' terminus containing the replication origin of parvovirus replicative form DNA.

The nucleotide sequence of the 5' terminus of the parvovirus H-1 was determined. There are two orientations of the 242-base-pair terminal palindrome in native replicative form DNA, one inverted with respect to the other. Adjacent to the terminal palindrome is an AT-rich region that is noncoding and contains a 55-base-pair tandem repeat. The addition mutant of H-1, DI-1, was also sequenced in this region and shown to have three copies of the tandem repeat sequence. Similarly, the related parvovirus H-3 contains only one copy of this repeat sequence. This region contains the replication origin for parvovirus replicative form DNA replication. Some of the implications of these results are discussed.