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Published December 2, 2021 | Accepted Version + Supplemental Material
Journal Article Open

Error-prone, stress-induced 3′ flap–based Okazaki fragment maturation supports cell survival


How cells with DNA replication defects acquire mutations that allow them to escape apoptosis under environmental stress is a long-standing question. Here, we report that an error-prone Okazaki fragment maturation (OFM) pathway is activated at restrictive temperatures in rad27Δ yeast cells. Restrictive temperature stress activated Dun1, facilitating transformation of unprocessed 5′ flaps into 3′ flaps, which were removed by 3′ nucleases, including DNA polymerase δ (Polδ). However, at certain regions, 3′ flaps formed secondary structures that facilitated 3′ end extension rather than degradation, producing alternative duplications with short spacer sequences, such as pol3 internal tandem duplications. Consequently, little 5′ flap was formed, suppressing rad27Δ-induced lethality at restrictive temperatures. We define a stress-induced, error-prone OFM pathway that generates mutations that counteract replication defects and drive cellular evolution and survival.

Additional Information

© 2021 American Association for the Advancement of Science. Received 20 April 2021; accepted 14 October 2021. We thank R. Kolodner for the yeast strains RDKY2672, RDKY2608, and RDKY2669; P. M. J. Burgers for the plasmids pBL335 (GST-Pol3), pBL338 (GAL1-Pol31), pBL340 (GAL10-Pol32), and pBL341 (Pol31/Pol32); L. Prakash and S. Prakash for the protease-deficient yeast strain YRP654 and the plasmids pBJ1445 (Flag-Pol3) and pBJ1524 (GST-Pol31/Pol32) to express the yeast recombinant Polδ complex (Pol3, Pol31, and Pol32); W.-D. Heyer for the anti-Dun1 antibody; and M. S. Wold for purified recombinant yeast replication protein A (RPA) complex. We thank H. Lou's laboratory members and H. Dai, D. Duenas, and M. E. Budd for technical assistance in mouse and yeast genetic experiments and stimulating discussions. We thank K. Walker and S. Wilkinson for critical reading and editing of the manuscript. This work was supported by NIH grants R50 CA211397 to L.Z. and R01 CA073764 and R01 CA085344 to B.S. Research reported in this publication includes work performed by City of Hope shared resources supported by the National Cancer Institute of the NIH under award number P30 CA033572. Author contributions: H.S., Z.L., A.S., Y.Z., and M.Z. conducted yeast genetic and biochemical experiments. E.Z., J.W., X.W., Z.H., and Z.G. conducted RNA sequencing (RNA-seq), WES, and WGS and performed data analysis. J.L.C. designed yeast genetic experiments and conducted data analysis. L.Z. conducted biochemical experiments, RNA-seq, and WGS data analysis; designed and coordinated most of the experiments; and wrote the first draft of the manuscript. B.S. supervised the entire project; designed and coordinated most of the experiments; and provided input into and finalized the manuscript. The authors declare no conflicts of interest in this study. Data and materials availability: All data are available in the manuscript or the supplementary materials. Gene Expression Omnibus accession numbers for the mouse and yeast genomics datasets are GSE181154 and GSE178876, respectively.

Attached Files

Accepted Version - nihms-1777671.pdf

Supplemental Material - science.abj1013_mdar_reproducibility_checklist.pdf

Supplemental Material - science.abj1013_sm.pdf


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Additional details

August 22, 2023
December 22, 2023