Project description:The eukaryotic GINS complex has an essential role in the initiation and elongation phases of genome duplication. It is composed of four paralogous subunits--Sld5, Psf1, Psf2 and Psf3--which are ubiquitous and evolutionarily conserved in eukaryotic organisms. Here, we report the biochemical characterization of the human GINS complex (hGINS). The four hGINS subunits were coexpressed in Escherichia coli in a highly soluble form and purified as a complex. hGINS was shown to interact directly with the heterodimeric human DNA primase, by using either surface plasmon resonance measurements or by immunoprecipitation experiments carried out with anti-hGINS antibodies. The DNA polymerase alpha-primase synthetic activity was specifically stimulated by hGINS on various primed DNA templates. The significance of these findings is discussed in view of the molecular dynamics at the human replication fork.

| S-EPMC1796748 | biostudies-literature

Movies for human polymerase alpha-primase in DNA elongation states

Project description: Not available

| EMPIAR-11361 | biostudies-other

Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase.

Project description:DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ~20 nucleotides of DNA. Here we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.

| S-EPMC10418221 | biostudies-literature

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