DNA Damage-Induced, S-Phase Specific Phosphorylation of Orc6 is Critical for the Maintenance of Genome Stability

Abstract

The smallest subunit of the human Origin Recognition Complex, hOrc6, is required for DNA replication progression and plays an important role in mismatch repair (MMR) during S-phase. However, the molecular details of how hOrc6 regulates DNA replication and DNA damage response remain to be elucidated. Orc6 levels are elevated upon specific types of genotoxic stress, and it is phosphorylated at Thr229, predominantly during S-phase, in response to oxidative stress. Many repair pathways, including MMR, mediate oxidative DNA damage repair. Defects in MMR are linked to Lynch syndrome, predisposing patients to many cancers, including colorectal cancer. Orc6 levels are known to be elevated in colorectal cancers. Interestingly, tumor cells show reduced hOrc6-Thr229 phosphorylation compared to adjacent normal mucosa. Further, elevated expression of wild-type and the phospho-dead forms of Orc6 results in increased tumorigenicity, implying that in the absence of this “checkpoint” signal, cells proliferate unabated. Based on these results, we propose that DNA-damage-induced hOrc6-pThr229 phosphorylation during S-phase facilitates ATR signaling in the S-phase, halts fork progression, and enables assembly of repair factors to mediate efficient repair to prevent tumorigenesis. Our study provides novel insights into how hOrc6 regulates genome stability.

Keywords:

• DNA damage
• Orc6
• phosphorylation
• replication

SUPPLEMENTAL MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2023.2196204.

ACKNOWLEDGEMENTS

We thank members of the Prasanth laboratory for discussions and suggestions. We thank Drs M. Aladjem, R. Bhargava, J. Cook, A. Dutta, H. Ducoff (posthumous), R. Hsu, M. Mechali, B. Moriarity, S. Nair, B. Stillman, M. Sverdlov, Y. Wang, M. Wold, L. Zou for providing reagents and insightful suggestions. This work was supported by NIH [R35 GM 122569] and NSF [PHY 1430124] awards to TH; NIH [R21AG065748; R01GM132458], Cancer center at Illinois seed grant and Prairie Dragon Paddlers awards [MCB1723008] to KVP; and NSF [1243372, 1818286, 2225264], NIH [GM125196] and CCIL awards to SGP. TH is an investigator with Howard Hughes Medical Institute.

AUTHOR CONTRIBUTIONS

Y-C.L., D.L. and A.C. designed, performed, and analyzed most experiments. J.S. helped with IP experiments; J.M. did SiMPull experiments; V.M, E.B, and A.K-B. did the IHC staining and analyses; L.S. did the invasion and migration experiment in M4 cells; T.H. provided technical support and conceptual advice toward SiMPull experiments. S.G.P. and K.V.P. supervised the project. Y-C.L., D.L., and S.G.P. wrote the manuscript.

DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are available within the article and in the supplementary material. No new datasets were generated in this study.