Abstract
The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2-7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2-7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.
Highlights
Sld3/Treslin are required for the initiation of DNA replication
The C-terminal Region of Sld3 Binds to Mcm3, Mcm5, and single-stranded DNA (ssDNA)—We previously found that Sld3 competes with GINS for the Mcm2–7 interaction with purified proteins [42]
Because it was determined by others that GINS binds to the Mcm3 and Mcm5 subunits of Mcm2–7 [15], we hypothesized that Sld3 may bind to Mcm3 and/or Mcm5
Summary
Sld3/Treslin are required for the initiation of DNA replication. Results: Sld interaction with ssDNA is required for GINS attachment to Mcm in yeast cells. The replication fork helicase is comprised of CMG (Cdc, Mcm, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. A second key event in helicase assembly is the attachment of GINS to the Mcm and Mcm subunits of the Mcm complex during S phase [15, 17, 35, 36]. Sld3/Treslin Regulates Helicase Assembly vitro, but that single-stranded DNA (ssDNA) releases these proteins from Mcm (40 – 44). Treslin binds to ssDNA, and ssDNA releases Treslin from Mcm and Mcm, analogous to the situation for yeast These results suggest a conserved mechanism for replication fork helicase assembly during S phase, governed by the Sld3/Treslin proteins
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