Functional analysis of the MCM2-7 DNA entry/exit gate during S-phase
General Genetics and Functional Genome Biology
Biochemistry
Final Report Abstract
Precise DNA replication is essential for all living organisms. Thus, eukaryotic cells have evolved a sophisticated system for replicating DNA. In the first step, the replicative helicase, MCM2-7, is loaded around double-stranded origin DNA (dsDNA) as a hexameric ring. In the second step, following a poorly understood process, MCM2-7 becomes activated in early S phase resulting in the establishment of bidirectional replication forks. Since MCM2-7 gets loaded on dsDNA but its active form encircles single-stranded DNA (ssDNA), regulated MCM2-7 ring opening and closing is crucial for its function and must happen during DNA replication initiation. The project aimed to uncover how regulated opening of the replicative helicase MCM2- 7 ring affects firing of replication origins and DNA synthesis in vivo. Using an exclusively available, chemical biology approach to control MCM2-7 DNA insertion/extrusion, the following aims were tackled: 1. Identification of the MCM2-7 ssDNA exit gate; 2. Characterisation of the replicative helicase activation process and 3. Highresolution mapping of the genome-wide binding of key replicative factors. To identify the ssDNA exit gate within MCM2-7 in vivo, FRB and FKBP protein domains were engineered onto neighbouring Mcm subunits in order to dimerise and form a rapamycindependent blockage. The previously described Mcm2/5 gate for loading MCM2-7 onto DNA was the only construct showing a severe growth and S phase initiation defect. These results, together with DNA synthesis data, chromatin binding analyses and ChIP-qPCR data defined the Mcm2/5 gate as the unique DNA entry and exit gate of the MCM2-7 replicative helicase. To further characterise the helicase, primed for DNA extrusion, we tested several key replication factors for association employing ChIP-qPCR, including DDK, Sld3, Cdc45, Dpb11, Sld2, Sld5, Mcm10, RPA, and Pol ε. We observed that when ssDNA could not be extruded, mainly Cdc45 and Sld3 stably associated with the helicase during S phase initiation. This and other data led us to the conclusion that production of ssDNA is an early key event in the activation of the inactive helicase. Only after initial unwinding/ extrusion of DNA from the N- terminal tier of MCM2-7, the helicase is properly maturated, as evaluated from ChIP-based assays. Finally, the introduction of high-resolution, genome-wide sequencing approaches to the lab (ChIP-Exo, ChIP-Seq, MNase-Seq and ssDNA-Seq) allowed us for the first time to gain a deeper insight into MCM2-7 location and activation on origin DNA in vivo. Our data suggests that ssDNA extrusion and initial DNA unwinding happens at the interface of the MCM2-7 double hexamer over the A-element of the ORC-binding site before the helicase is fully matured into a CMG complex. Then, helicase is initiated by Mcm10 and CMGs pass each other to establish a pair of diverging replication forks.
