Activity, substrate preference and structure of the HsMCM8/9 helicase

Abstract

The minichromosomal maintenance proteins, MCM8 and MCM9, are more recent evolutionary additions to the MCM family, only cooccurring in selected higher eukaryotes. Mutations in these genes are directly linked to ovarian insufficiency, infertility, and several cancers. MCM8/9 appears to have ancillary roles in fork progression and recombination of broken replication forks. However, the biochemical activity, specificities and structures have not been adequately illustrated, making mechanistic determination difficult. Here, we show that human MCM8/9 (HsMCM8/9) is an ATP dependent DNA helicase that unwinds fork DNA substrates with a 3′–5′ polarity. High affinity ssDNA binding occurs in the presence of nucleoside triphosphates, while ATP hydrolysis weakens the interaction with DNA. The cryo-EM structure of the HsMCM8/9 heterohexamer was solved at 4.3 Å revealing a trimer of heterodimer configuration with two types of interfacial AAA+ nucleotide binding sites that become more organized upon binding ADP. Local refinements of the N or C-terminal domains (NTD or CTD) improved the resolution to 3.9 or 4.1 Å, respectively, and shows a large displacement in the CTD. Changes in AAA+ CTD upon nucleotide binding and a large swing between the NTD and CTD likely implies that MCM8/9 utilizes a sequential subunit translocation mechanism for DNA unwinding.

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 .…”

“…The experiments presented in Fig. 2f clearly support the scenario 1 and thus the 3’-5’ translocation polarity, in agreement with data obtained with truncated MCM8-9 without HROB previously 18 . The observed 3’-5’ translocation polarity of MCM8-9 is consistent with that of replicative MCM proteins from archaea or eukaryotes (MCM2-7) 46 .…”

“…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 .…”

“…2e,f ). We reasoned that the very short flaps (5 nt) are unlikely to allow the loading of MCM8-9; in fact, the truncated MCM8-9 complex failed to load on 10-nt-long overhangs in a previous study 18 . We anticipated that MCM8-9 instead loads onto and translocates along the circular ssDNA, but the direction of translocation was unknown.…”