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Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

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Specific origin selection and excess functional MCM2-7 loading in ORC-deficient cells.

Yoshiyuki Shibata¹, Mihaela Peycheva², Etsuko Shibata¹

¹Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, United States.

Nucleic Acids Research

|June 18, 2025

Summary

The six-subunit origin recognition complex (ORC) is not essential for DNA replication origin specification or MCM2-7 loading in human cancer cells. Specific origins are still used and excess MCM2-7 is loaded even without ORC.

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Area of Science:

Molecular Biology
Genetics
Cell Biology

Background:

The origin recognition complex (ORC) is crucial for initiating DNA replication by loading MCM2-7 onto chromosomes.
ORC is thought to play a key role in specifying DNA replication origins.

Purpose of the Study:

To investigate the necessity of the six-subunit ORC for origin specification and MCM2-7 loading in human cancer cell lines.
To determine if specific origins are maintained and if excess MCM2-7 loading occurs in the absence of ORC subunits.

Main Methods:

Engineered human cancer cell lines with deletions of ORC1, ORC2, or ORC5 subunits.
Mapped DNA replication origins in these engineered cell lines.
Assessed MCM2-7 loading rates during G1 and S phases.

Main Results:

Specific DNA replication origins were utilized at similar genomic sites in ORC-deficient cells compared to wild-type cells.
GC/TA skewness and simple repeats were found to facilitate, but not be essential for, origin selection without ORC.
Excess MCM2-7 was loaded at comparable rates in G1 phase and re-loaded in S phase, indicating licensing of dormant origins and permitting re-replication despite ORC absence.

Conclusions:

The six-subunit ORC is not required for origin specification in human cancer cell lines.
Excess MCM2-7 loading and licensing of dormant origins occur independently of the six-subunit ORC.
These findings challenge the established role of ORC in origin selection and MCM loading.