Abstract
The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms.
Highlights
Living organisms have evolved different strategies to recruit and load DNA helicases at the replication origins, and accessory factors, referred to as helicase loaders, are required to accomplish this task [1, 2]
DNA Binding Activity of SsoCdc6-2 and SsoMCM—The ability of SsoCdc6-2 to bind DNA in a structure-dependent manner was assayed using a standard electrophoretic mobility shift assay on a variety of synthetic oligonucleotides: molecules containing a bubble of 20 T residues (Bub-20T), flayed duplexes with tails of 20 T residues (Fork), 3Ј- or 5Ј-tailed duplexes (3Ј-Tail and 5Ј-Tail), blunt double-stranded DNA molecules, and single-stranded DNA oligonucleotides
Because SsoMCM hexamers are not stable and dissociate upon dilution [12], it is likely that the faster migrating bands are produced by binding to DNA of these various SsoMCM subassemblies, whereas the slower migrating complex found at the higher protein concentrations could contain one or possibly two SsoMCM hexamers
Summary
Proteins—SsoMCM, wild type and KA mutant SsoCdc, and SsoCdc6-2 ⌬C were purified as described [12,13,14]. The production of SsoMCM ⌬268 will be published elsewhere.
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