Structural and mechanistic insights into the MCM8/9 helicase complex

Abstract

MCM8 and MCM9 form a functional helicase complex (MCM8/9) that plays an essential role in DNA homologous recombination repair for DNA double-strand break. However, the structural characterization of MCM8/9 for DNA binding/unwinding remains unclear. Here, we report structures of the MCM8/9 complex using cryo-electron microscopy single particle analysis. The structures reveal that MCM8/9 is arranged into a heterohexamer through a threefold symmetry axis, creating a central channel that accommodates DNA. Multiple characteristic hairpins from the N-terminal oligosaccharide/oligonucleotide (OB) domains of MCM8/9 protrude into the central channel and serve to unwind the duplex DNA. When activated by HROB, the structure of MCM8/9’s N-tier ring converts its symmetry from C3 to C1 with a conformational change that expands the MCM8/9’s trimer interface. Moreover, our structural dynamic analyses revealed that the flexible C-tier ring exhibited rotary motions relative to the N-tier ring, which is required for the unwinding ability of MCM8/9. In summary, our structural and biochemistry study provides a basis for understanding the DNA unwinding mechanism of MCM8/9 helicase in homologous recombination.

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

“…9c-f ). These results reinforce the notion that MCM8-9 unwinds DNA as a hexamer, which is further supported by our AlphaFold2 modeling and recent structural studies 18 , 20 .…”

“…We employed a combination of structure prediction, mutagenesis and ensemble as well as single-molecule biochemical approaches to gain insights into the mechanism of DNA unwinding by MCM8-9 and its regulation by HROB. We demonstrate that MCM8 and MCM9 proteins in solution form diverse oligomeric species ranging from dimers to hexamers, in agreement with recent structural studies focused on chicken MCM8-9 20 or truncated human MCM8-9 complex 18 . Our biochemical data suggest that MCM8-9 hexamers are necessary for DNA unwinding in conjunction with HROB.…”

“…AlphaFold2 predicted that MCM8-9 forms a hexamer (Fig. 1i ), which is very similar to the recent cryoEM structure obtained with human MCM8-9 N-terminal domains 19 , chicken MCM8-9 20 , or truncated human MCM8-9 complex 18 . The structural model predicts one molecule of HROB to bind both subunits of the MCM8-9 heterodimer (Fig.…”

“…4e ). The mean diameter of the clearly visible MCM8-9 rings in the negative staining transmission electron microscopy images was 14 nm, which is similar to the mean ring diameter measured in cryoEM images of full-length chicken MCM8-9 hexamer (13.2 nm) 20 , truncated human MCM8-9 (14 nm) 18 or the MCM2-7 hexamer from budding yeast (13.3 nm) 52 .…”

“…They are missing in fungi and nematodes, but present in most plants and vertebrates, while Drosophila only contains an MCM8 homologue 14 – 16 . The MCM8 and MCM9 proteins associate together (MCM8-9) and are similarly thought to form hexamers 17 – 20 , yet their assembly, function and mode of activation remain poorly understood. While MCM8 has the standard domain structure, MCM9 contains an unusual C-terminal extension downstream of the AAA+ ATPase site 21 .…”