Latest Research

[PRESS RELEASE] Mechanism of Replicative Helicase Activation in DNA Replication

　Kanemaki Group / Molecular Cell Engineering Laboratory
Toyoda Group / Comparative Genomics Laboratory

MCM10 and RECQL4 have cooperative and redundant roles in activating the CMG helicase during the replication initiation

Atabek Bektash, Xiaoxuan Zhu, Yuki Hatoyama, Atsushi Toyoda and Masato T. Kanemaki

Genes & Development　DOI:10.1101/gad.353714.126

Press Release

For the cells that make up our bodies to proliferate, they must accurately copy the DNA that carries their genetic information through a process known as DNA replication. At the onset of DNA replication, a molecular motor called the CMG helicase is assembled to unwind double-stranded DNA. DNA replication begins when this helicase is activated. However, the mechanism by which the CMG helicase is activated in human cells has remained poorly understood.

In this study, we used an improved auxin-inducible degron (AID2) technology that we have developed to rapidly remove two proteins involved in DNA replication, MCM10 and RECQL4, from human cells and investigate their roles. We found that depletion of either protein alone had a relatively mild effect on DNA replication. In contrast, simultaneous depletion of both proteins severely impaired DNA replication. Although the CMG helicase could still be assembled, it failed to become activated. These results indicate that MCM10 and RECQL4 are involved in CMG activation (Figure A).

Figure 1: Schematic illustration of DNA replication initiation in human cells. First, two ring-shaped MCM complexes are loaded onto DNA to prepare for replication (licensing). Subsequently, through the action of multiple replication factors, GINS and CDC45 associate with MCM to form the CMG helicase (CMG assembly). Finally, MCM10 and RECQL4 promote activation of the CMG helicase (CMG activation).

Further analyses revealed that MCM10 and RECQL4 not only cooperate with each other but can also partially compensate for the loss of the other protein. In particular, we found that the ability of both proteins to bind single-stranded DNA is important for CMG helicase activation.

Based on these findings, we propose a new model in which MCM10 and RECQL4 act cooperatively and redundantly to activate the CMG helicase by promoting the extrusion of single-stranded DNA from the helicase (Figure B). This study advances our understanding of the fundamental mechanism by which DNA replication is initiated in human cells.

Figure 2: MCM10 and RECQL4 interact with each other and cooperate to promote CMG helicase activation. Both proteins contain single-stranded DNA (ssDNA)-binding domains. We propose that MCM10 and RECQL4 use their ssDNA-binding activity to promote the extrusion of ssDNA from the ring-shaped CMG helicase, thereby activating the helicase.

This work was supported by JSPS KAKENHI grants (JP21H04719, JP23H04925 and JP25H00979) and JST CREST (JPMJCR21E6). Genomic analyses were supported by the Platform for Advanced Genome Science (PAGS, JP22H04925) and the NIG Supercomputer Facility. Atabek Bektash conducted this research as a Japanese Government (MEXT) Scholarship student.

Glossary

Auxin-Inducible Degron (AID) Technology:
A protein degradation technology developed by the Kanemaki laboratory. It was created by transferring the auxin-dependent protein degradation system found in plants into non-plant cells. The improved AID2 system has been applied to a wide range of cells and organisms, including yeast, cultured cells, C. elegans, and mice.

CMG helicase:
A molecular machine that unwinds double-stranded DNA into single-stranded DNA during DNA replication. By generating single-stranded DNA, the CMG helicase allows DNA polymerases to synthesize new DNA strands.

MCM10:
A protein involved in DNA replication that is conserved from yeast to humans. Until now, how human MCM10 contributes to DNA replication has remained poorly understood.

RECQL4:
A protein thought to be involved in DNA replication in human cells, although its precise role in this process has remained poorly understood. Mutations in RECQL4 are known to cause genetic disorders, including Rothmund–Thomson syndrome.