In eukaryotes, during G1 phase, the ‘pre-replication complex’ is formed at the origin of replication comprised of origin recognition complex (ORC), Cdc6, Cdt1 (chromatin licensing and DNA replication factor) and minichromosome maintenance proteins Mcm2-7. In S phase, phosphorylation by cyclin dependent kinase 2 (CDK2) and DDK activates the complex to transit to the ‘initiation complex’. Mcm2-7 forms double hexamer rings on DNA that serves as scaffold for the assembly of ‘CMG’ (cdc45-MCM-GINS) helicase and the replisome complex (Parker et al., 2016). Intriguingly, Mcm7 has been shown to associate with the tumor suppressor Rb linked to retinoblastoma/osteosarcoma and mutated in ~30% of human cancers including non-small cell lung cancer, prostate cancer, glioma and breast cancer (Sterner et al.., 1998; Bookstein et al., 1990). Cdc45 loads DNA polymerase, PCNA, RPA and others. GINS and Cdc45 augment the helicase activity.
The exact mechanism through which bi-directional replication forks are generated is not clearly understood. Previous works have shown that the two head-to-head CMG complexes assembled on dsDNA undergo separation, and, upon binding to Mcm10, both complexes engage opposite single strands of the unwound DNA and go past one another to form bi-directional forks. However, whether CMG and Mcm10 can untwist double helix sufficiently to allow CMG to translocate to ssDNA is not known.
To determine if the ring-shaped CMG motor could force open duplex of sufficient length, researchers (Rockefeller University) designed a T-shaped dsDNA probe, which allows CMG to load and slide but will encounter a block at the T-junction. Here, the ability of CMG to melt the DNA would allow a labeled oligonucleotide annealed at the T-junction to dissociate for monitoring. By varying the length of DNA at the T-junction, they determined that CMG (plus Mcm10) could unwind 60-bp dsDNA (Fig 3 inLangston et al., 2019).
Next, to determine how it opens dsDNA, either strand of the probe (where CMG slides before the T-junction) was partly replaced with 10 neutral methylphosphonate linkages acquired from Bio-Synthesis, Inc. The duplex unwinding was significantly reduced when the methylphosphonate linkage was incorporated on the 3’-5’ strand in the direction of unwinding, suggesting that CMG tracks along this strand primarily while encircling dsDNA (Langston et al., 2019). Subsequent experiments corroborated the above finding, suggesting that the head-to-head CMG complexes plus Mcm10 generate sufficient force to open several turns of the duplex.
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References
Bookstein R, Rio P, Madreperla SA, Hong F, Allred C, Grizzle WE, Lee WH. Promoter deletion and loss of retinoblastoma gene expression in human prostate carcinoma. (1990) Proc Natl Acad Sci USA 87:7762-6. PMID: 2217208 PMCID: PMC54828 DOI: 10.1073/pnas.87.19.7762
Kaguni JM. Replication initiation at the Escherichia coli chromosomal origin. (2011) Curr Opin Chem Biol 15: 606–13. PMC 3189269 PMID 21856207 doi:10.1016/j.cbpa.2011.07.016
Langston LD, O'Donnell ME. An explanation for origin unwinding in eukaryotes. (2019) Elife. Jul 8;8. pii: e46515. PMID: 31282859 doi: 10.7554/eLife.46515.
Parker MW, Botchan MR, Berger JM. Mechanisms and regulation of DNA replication initiation in eukaryotes. (2017) Crit Rev Biochem Mol Biol 52:107-144. PMID: 28094588 doi: 10.1080/10409238.2016.1274717
Sterner JM, Dew-Knight S, Musahl C, Kornbluth S, Horowitz JM. Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7. (1998) Mol Cell Biol 18:2748-57. PMID: 9566894 PMCID: PMC110654 DOI: 10.1128/mcb.18.5.2748