Mechanism of DNA unwinding by MCM8-9 in complex with HROB

HROB promotes the MCM8-9 helicase in DNA damage response. To understand
how HROB activates MCM8-9, we defined their interaction interface. We
showed that HROB makes important yet transient contacts with both MCM8 and
MCM9, and binds the MCM8-9 heterodimer with the highest affinity.
MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding
processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers
on DNA in the presence of ATP. The hexamer involves two repeating
protein-protein interfaces between the alternating MCM8 and MCM9 subunits.
One of these interfaces is quite stable and forms an obligate heterodimer
across which HROB binds. The other interface is labile and mediates
hexamer assembly, independently of HROB. The ATPase site formed at the
labile interface contributes disproportionally more to DNA unwinding than
that at the stable interface. Here, we show that HROB promotes DNA
unwinding downstream of MCM8-9 loading and ring formation on ssDNA.