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ACKNOWLEDGMENTS
Funding:K.D.B and D.J. are supported by NSF grant IOS-1934388.
K.D.B, D.J., and T.R.G. were supported by NSF grant IOS 1445025.
K.D.B. is funded by NIH grant R35GM136362. D.J. is funded by
NSF IOS-1930101. K.L.G. is funded by NSF PGRP-23020. B.G. is
funded by the Human Frontier Science Program Organization
grant: LT - 000972/2018.Author contributions:C.O.-R., B.G.,
S.Z., and L.L. performed all transcriptomic experiments and
expression analysis. C.O.R. performed mutant analysis. C.O.R. and
K.D.B. designed the experiments and wrote the manuscript. C.O.R.,
K.D.B., D.J., T.R.G., and K.L.G. conceived the project and guided
the experiments. X.X. performed in situ hybridizations. P.C.D.A.
and S.Z. assisted in mutant analysis and marker analysis. R.R.
designed and carried out microscopy protocols and designed
graphic layouts. E.D.-A. and X.X. generated transcriptional reporters
in maize. C.O.R., Z.Y., and J.V.E generated CRISPR-Cas9 knockouts
and translational reporters in maize andSetariaand generatedtheSetariamutants.Competing interests:The authors declare
no competing interests.Data materials and availability:All
data are included in the main paper or the supplementary
materials or are deposited at the Gene Expression Omnibus
website (https://www.ncbi.nlm.nih.gov/geo/) under the
SuperSeries accession number GSE172302.pZmWOX5::RFP,
pZmSCR1::RFP, andpZmSHR2::RFPreporter lines are available
from D.J. under a material transfer agreement with Cold Harbor
Spring Laboratory.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abj2327
Materials and Methods
Figs. S1 to S13
Tables S1 to S3
References ( 24 Ð 30 )
MDAR Reproducibility Checklist28 April 2021; accepted 13 October 2021
10.1126/science.abj2327REPORTS
◥MOLECULAR BIOLOGY
Error-prone, stress-induced 3′flap–based Okazaki
fragment maturation supports cell survival
Haitao Sun^1 , Zhaoning Lu^1 , Amanpreet Singh^1 , Yajing Zhou^1 , Eric Zheng1,2, Mian Zhou^1 ,
Jinhui Wang^3 , Xiwei Wu^3 , Zunsong Hu^4 , Zhaohui Gu^4 , Judith L. Campbell^5 ,
Li Zheng^1 , Binghui Shen^1
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 inrad27Dyeast cells. Restrictive
temperature stress activated Dun1, facilitating transformation of unprocessed 5′flaps into 3′flaps,
which were removed by 3′nucleases, including DNA polymerased(Pold). 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 aspol3internal tandem duplications.
Consequently, little 5′flap was formed, suppressingrad27D-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.
U
nderstanding the mutagenesis mech-
anisms that are active in cells under
stress conditions is crucial for develop-
ing strategies to intervene in microbial
pathogenesis, tumorigenesis, and drug
resistance ( 1 , 2 ). Lagging-strand DNA synthe-
sisisparticularlyvulnerabletostressand
environmental factors. During replication,
lagging-strand DNA is synthesized as dis-
crete Okazaki fragments ( 3 ), which contain
short primase- and DNA polymerasea(Pola)–
synthesized RNA-DNA primers at their 5′ends
as well as the DNA fragment that is extended
by DNA polymerased(Pold)( 4 – 6 ). During
Okazaki fragment maturation (OFM), the RNA
portion and any Pola-synthesized DNA with a
high number of incorporation errors are dis-
placed through Pold-mediated DNA synthesis,
which produces a 5′RNA-DNA flap ( 4 – 6 ). The
5 ′flap structure is removed by flap endonu-
clease1(FEN1)orthroughthesequential
actions of DNA2 nuclease-helicase and FEN1
( 7 – 9 ). FEN1 deficiency leads to the accumula-
tion of unprocessed 5′flap structures, which
may prevent ligation of Okazaki fragments,leaving DNA nicks or gaps that lead to the
collapse of replication forks and DNA double-
strand breaks. In yeast, deletion of the FEN1
homologRAD27(rad27D) results in slow growth
at permissive growth temperatures (30°C)
and death at restrictive growth temperatures
(37°C) ( 10 ).
Nevertheless, we discovered that a small pop-
ulation ofrad27Dyeast cells, which we called
revertants, could grow at a similar rate as wild-
type (WT) cells at 37°C (Fig. 1A). To determine
if the revertants acquired somatic mutation(s)
that permitted growth and to identify any such
mutation(s), we conducted whole-genome se-
quencing (WGS) of WT, parentalrad27D, and
a revertant strain of yeast cells. We identified
21 somatic DNA mutations specific to one re-
vertant colony (table S1). A mutation inPOL3,
the Poldcatalytic subunit ( 11 ), was the only
nonsynonymous mutation that had 100% al-
lele frequency in the revertant. Subsequent
polymerase chain reaction (PCR)–based DNA
sequencing analysis of thePOL3gene in inde-
pendentrad27Drevertant colonies (n= 31)
revealed that each revertant colony harbored
apol3mutation (Fig. 1B). This suggested that
thesepol3mutations, which mapped onto
Pol3 functional motifs (Fig. 1B and supple-
mentary text S1) and possibly affected its
biochemical activities, might provide a survi-
val advantage forrad27Dcells grown under
restrictive temperature stress. Furthermore,
knock in of the 458–477 internal tandem
duplication (ITD) mutation, which occur-
red in 19 of the 31 independent colonies, or
any of the four representative point mutations
[Arg^470 →Gly (R470G), Arg^475 →Ile (R475I),
Ala^484 →Val (A484V), and Ser^847 →Tyr (S847Y)]
successfully reversed the restrictive temperature–
induced lethality phenotype ofrad27Dcells
(Fig. 1C and fig. S1).rad27Dcells are sensitive
to methyl methanesulfonate (MMS) ( 10 ). Al-
thoughrad27Drevertant cells andrad27Dpol3
ITD knock-in mutant cells were resistant to1252 3 DECEMBER 2021•VOL 374 ISSUE 6572 science.orgSCIENCE
(^1) Department of Cancer Genetics and Epigenetics, Beckman
Research Institute, City of Hope, 1500 East Duarte Road,
Duarte, CA 91010, USA.^2 Department of Molecular, Cellular,
and Developmental Biology, University of California at Santa
Barbara, Santa Barbara, CA 93106, USA.^3 Department of
Molecular and Cellular Biology, Beckman Research Institute,
City of Hope, 1500 East Duarte Road, Duarte, CA 91010,
USA.^4 Department of Computational and Quantitative
Medicine, Beckman Research Institute, City of Hope, 1500
East Duarte Road, Duarte, CA 91010, USA.^5 Braun
Laboratories, California Institute of Technology, Pasadena,
CA 91125, USA.
*Corresponding author. Email: [email protected] (L.Z.); bshen@
coh.org (B.S.)
RESEARCH