Abstract
The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3′ protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3′–5′ polarity and, thereby acts as a molecular ‘wedge’ to initiate DNA strand displacement.
Highlights
Faithful duplication of eukaryotic DNA requires the ordered and highly coordinated recruitment of many proteins into the origins of replication to form a replication initiation complex [1,2,3,4,5]
Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that human Cdc45 (hCdc45) exists as an alphahelical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 30 protruding strands and bound tightly to single-strand/doublestrand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication
The quality of the purified protein was controlled by dynamic light scattering, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), all of which indicated a monodisperse behavior
Summary
Faithful duplication of eukaryotic DNA requires the ordered and highly coordinated recruitment of many proteins into the origins of replication to form a replication initiation complex [1,2,3,4,5]. Compared with Mcm alone, the CMG complex exhibits a much stronger DNA helicase and ATPase activity [15], indicating that Cdc and GINS are true subunits of the replicative DNA helicase This was confirmed by single-particle electron microscopy studies showing that the Mcm complex forms an open lockwasher structure, and binding of Cdc and GINS causes a closed-ring formation [16]. This conformational change is mandatory to efficiently activate the helicase function of CMG [15]. HCdc seems to slide along ssDNA with a 30–50 polarity until it reaches an ss/double-strand DNA (dsDNA) junction, consistent with a wedge function of Cdc within the CMG complex
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