Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)

Abstract

The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea͞ eukaryotes. Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities. Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif. We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC. In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes.T he universality of the processes involved in the transmission of genetic information has been interpreted to reflect a common evolutionary history. Thus it is accepted that life originated from self-replicating RNA molecules capable of directing protein synthesis, to be replaced later by DNA as the genetic material (1). However, this view has been challenged by modern genomics, inasmuch as protein sequence comparisons conclude that the set of genes for replication, transcription, and translation in bacteria differs from that found in archaea and eukarya (2). Divergence is noteworthy for proteins that initiate chromosomal DNA replication in bacteria (DnaA) (3) and eukarya (the six subunits of the origin recognition complex, ORC) (4, 5); despite their common function in binding to DNA replicators, they lack significant sequence similarity, other than an AATϩ module for ATP binding (6-8). However, structural similarities have been found among some of the accessory factors of prokaryotic and eukaryotic replicative DNA polymerases (9-11).We had previously found in RepA, the Pseudomonas plasmid pPS10 DNA replication initiator protein (12), the leucine-zipper (LZ) (13, 14) and helix-turn-helix (15) sequence motifs that function in protein dimerization and protein-DNA interaction, respectively. These motifs are part of two repeated winged-helix (WH) domains (16). In common with other homologous plasmid initiators (17, 18), RepA molecules exist as monomers and dimers in equilibrium (14). The monomers activate initiation of replication by binding to directly repeated DNA sequences (14, 16), whereas the dimers repress repA transcription by binding to an inversely repeated DNA operator (19,16). Dissociation of RepA dimers can either occur spontaneously (14,20) or be mediated by DnaK͞Hsp70 chaperones (21). Monomerization is coupled to a conformational change (22,23) in the N-terminal domain in RepA. As a result both the N-and C-terminal domains of the monomer show DNA binding, whereas in the dimer only the C-terminal domain is involved in DNA binding (16). This model was recent...

Extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes

“…This arrangement of two wHTH domains is reminiscent of the RepA plasmid initiator protein from the bacterium Pseudomonas. Significantly, structural similarities have previously been observed between bacterial RepA and archaeal Cdc6 initiator proteins (21 As can be seen in Fig. 5, Sso0867 binds the origin region producing a specific DNaseI footprint (Fig.…”

Structural Changes in RepA, a Plasmid Replication Initiator, upon Binding to Origin DNA

“…22). Furthermore, a similar WH fold is found at the C terminus of the eukaryotic/archaeal initiators Orc4/ Cdc6 (4,23), underlining the relevance of studying the molecular mechanism of RepA activation. We had proposed that dissociation of RepA dimers into monomers would result in a structural change altering the relative arrangement and compaction of its two WH domains (21).…”

“…Thermal denaturation analyses performed by optical techniques, such as CD spectroscopy, provide valuable information on the thermodynamic stability of proteins (4,21) and their complexes with DNA (55). We have carried out this kind of approach to study the effect on RepA stability of binding to the DNA sequences described above (Fig.…”

“…This is achieved by means of complex circuits that are distinct in prokaryotes and eukaryotes, although some similarities have become evident recently (2)(3)(4). Common essential elements are a cis-acting DNA sequence (the origin of replication) and a trans-acting factor (the initiator).…”

Sequential and Ordered Assembly of E1 Initiator Complexes on the Papillomavirus Origin of DNA Replication Generates Progressive Structural Changes Related to Melting

“…Until recently, little connection to initiators from higher organisms has been found. Recent studies, however, demonstrate that RepA, the initiator from a Pseudomonas plasmid is related to CDC6/ORC proteins, thus forging a link between the prokaryotic and eukaryotic families of initiator proteins (16). This provides a strong justification for understanding how the relatively low-complexity viral initiators perform their function since this would likely provide information about how DnaA, and possibly even initiators from higher organisms, function.…”