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1.
bioRxiv ; 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38370757

ABSTRACT

The selection of replication origins is a defining characteristic of DNA replication in eukaryotes, yet its mechanism in humans has not been well-defined. In this study, we use Cut&Run to examine genomic binding locations for TICRR/TRESLIN and MTBP, the human orthologs for the yeast DNA replication initiation factors Sld3 and Sld7. We mapped TRESLIN and MTBP binding in HCT116 colorectal cancer cells using asynchronous and G1 synchronized populations. Our data show that TRESLIN and MTBP binding patterns are more defined in a G1 synchronized population compared to asynchronously cycling cells. We also examined whether TRESLIN and MTBP are dependent on one another for binding. Our data suggest MTBP is dependent on TRESLIN for proper association with chromatin during G1 but not S phase. Finally, we asked whether TRESLIN and MTBP binding to chromatin requires licensed origins. Using cell lines with a non-degradable inducible Geminin to inhibit licensing, we show TRESLIN and MTBP binding does not require loaded MCMs. Altogether, our Cut&Run data provides evidence for a chromatin binding mechanism of TRESLIN-MTBP during G1 that is dependent on TRESLIN and does not require interactions with licensed origins.

2.
Nucleic Acids Res ; 49(18): 10507-10523, 2021 10 11.
Article in English | MEDLINE | ID: mdl-34534348

ABSTRACT

A DNA replication program, which ensures that the genome is accurately and wholly replicated, is established during G1, before the onset of S phase. In G1, replication origins are licensed, and upon S phase entry, a subset of these will form active replisomes. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the level of TICRR and its phosphorylation determine the number of origins that initiate during S phase. However, the mechanisms regulating TICRR protein levels are unknown. Therefore, we set out to define the TICRR/TRESLIN protein dynamics throughout the cell cycle. Here, we show that TICRR levels are high during G1 and dramatically decrease as cells enter S phase and begin DNA replication. We show that degradation of TICRR occurs specifically during S phase and depends on ubiquitin ligases and proteasomal degradation. Using two targeted siRNA screens, we identify CRL4DTL as a cullin complex necessary for TICRR degradation. We propose that this mechanism moderates the level of TICRR protein available for replication initiation, ensuring the proper number of active origins as cells progress through S phase.


Subject(s)
Cell Cycle Proteins/metabolism , S Phase , Ubiquitin-Protein Ligases/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , Carrier Proteins/physiology , Cell Cycle , Cell Cycle Proteins/physiology , Cell Line, Tumor , Cullin Proteins/metabolism , Cullin Proteins/physiology , Cyclin-Dependent Kinase 2/antagonists & inhibitors , DNA-Binding Proteins/physiology , Humans , Proliferating Cell Nuclear Antigen/physiology , Ubiquitin-Protein Ligases/physiology
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