Dpb11 helps control assembly of the Cdc45-Mcm2-7-GINS replication fork helicase [DNA and Chromosomes]

February 6th, 2015 by Dhingra, N., Bruck, I., Smith, S., Ning, B., Kaplan, D. L.

Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-CDK phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7 and GINS. We show here using purified proteins from budding yeast that Dpb11 alone binds to Mcm2-7, and Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased RPA interaction with origin DNA during S phase. We propose a model wherein Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, while bound to Cdc45-Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon the extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7 and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45-Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.