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 Zheng
1,2
,
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,*
1
Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope,
1500 East Duarte Road, Duarte, CA 91010
2
Department of Molecular, Cellular, and Developmental Biology, University of California at Santa
Barbara, Santa Barbara, CA 93106
3
Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1500
East Duarte Road, Duarte, CA 91010
4
Department of Computational and Quantitative Medicine, Beckman Research Institute, City of
Hope, 1500 East Duarte Road, Duarte, CA 91010
5
Divisions of Chemistry and Chemical Engineering and Biology and Biological Engineering
California Institute of Technology, Pasadena, CA 91125, USA
Abstract
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 an error-prone Okazaki
fragment maturation (OFM) pathway that is activated at restrictive temperatures in
rad27
Δ yeast
cells. Restrictive temperature stress activates Dun1, facilitating transformation of un-processed
5’ flaps into 3’ flaps, which are removed by 3’ nucleases including Pol
δ
. However, at certain
regions, 3’ flaps form secondary structures that facilitate 3’ end extension rather than degradation,
producing alternative duplications with short spacer sequences. Once such mutations occur at
POL3
, it fails to displace 5’flaps, thus rescues
rad27
Δ cells. Our study defines a stress-induced,
error-prone OFM pathway that generates mutations that counteract replication defects and drive
cellular evolution and survival.
Understanding the mutagenesis mechanisms that are active in cells under stress conditions
is crucial for developing strategies to intervene in microbial pathogenesis, tumorigenesis,
and drug resistance (
1
,
2
). Lagging-strand DNA synthesis is particularly vulnerable to
stress and environmental factors. During replication, lagging-strand DNA is synthesized as
*
Corresponding authors: lzheng@coh.org (L.Z.); bshen@coh.org (B.S.).
Author contributions:
H. Sun, Z. Lu, A. Singh, Y. Zhou, M. Zhou conducted yeast genetic and biochemical experiments. E. Zheng,
J. Wang, X. Wu, Z. Hu, and Z. Gu conducted RNA-seq, WES, and WGS and performed data analysis. J.L. Campbell designed yeast
genetic experiments and conducted data analysis. L. Zheng 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. Shen supervised the entire project,
designed and coordinated most of the experiments, and provided input into and finalized the manuscript.
Competing interests:
The authors declare no conflicts of interest in this study.
HHS Public Access
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Science
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discrete Okazaki fragments (
3
), which contain short primase/Pol
α
-synthesized RNA-DNA
primers at their 5’ ends (
4
-
6
). During Okazaki fragment maturation (OFM), the RNA
portion and any Pol
α
-synthesized DNA with high incorporation errors are removed, via
Pol
δ
-mediated displacement DNA synthesis, which produces a 5’ RNA-DNA flap (
4
-
6
).
The 5’ flap structure is removed by flap endonuclease 1 (FEN1) or through the sequential
actions of DNA2 nuclease/helicase and FEN1 (
7
-
9
). FEN1 deficiency leads to accumulation
of unprocessed 5’ flap structures, which may prevent ligation of Okazaki fragments, leaving
DNA nicks or gaps that lead to collapse of replication forks and DNA double-strand
breaks. In yeast, deletion of the FEN1 homolog
RAD27
(
rad27
)Δ 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 population of
rad27
Δ yeast 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 sequencing (WGS) of WT,
parental
rad27
Δ, and a revertant strain of yeast cells. We identified 21 somatic DNA
mutations specific to one revertant colony (Table S1). A mutation in
POL3
, the DNA
polymerase delta (Pol
δ
) catalytic subunit (
11
), was the only nonsynonymous mutation
that had 100% allele frequency in the revertant. Subsequent DNA sequencing analysis
of the
POL3
gene in independent
rad27
Δ revertant colonies (n = 31) revealed that each
colony harbored a
pol3
mutation (Fig. 1B). This suggests that these
pol3
mutations, which
map onto POL3 functional motifs (Fig. 1B, Supplementary text S1) and possibly affect
its biochemical activities, might provide a survival advantage for
rad27
Δ cells grown
under restrictive temperature stress. Furthermore, knock-in of the 458–477 internal tandem
duplication (ITD) mutation, which occurred in 19 of the 31 independent colonies, or any
of the four representative point mutations (R470G, R475I, A484V, and S847Y) successfully
reversed the restrictive temperature-induced lethality phenotype of
rad27
Δ cells (Fig. 1C
and fig. S1).
rad27
Δ cells are sensitive to methyl methanesulfonate (MMS) (
10
). Although
rad27
Δ revertant cells and
rad27
Δ
pol3
ITD knock-in mutant cells were resistant to a low
concentration (0.005%) of MMS, they were sensitive to higher concentrations (≥0.01%)
of MMS (fig. S2). We observed that
pol3
ITD cells in a WT RAD27 background were
also sensitive to high concentrations of MMS (fig. S2). This at least partially explains why
the
pol3
ITD could not suppress MMS-induced lethality of
rad27
Δ cells at high MMS
concentrations. In addition,
pol3
ITD did not rescue the synthetic lethality that occurs in
the context of
rad27
Δ coupled with deficiency of the 5’ nucleases
EXO1
or
DNA2
nuclease/
helicase (Tables S2, S3, Supplementary text S2).
Two types of duplications were present in the revertants:
pol3
591–598 ITD, a previously
reported classic duplication resulting from re-alignment and ligation of unprocessed 5’ flaps
(
12
), and
pol3
458–477 ITD, which contained a 55 bp duplication with a 5 bp spacer
between the duplicated units (fig. S3). We named the duplication with an intervening spacer
an “alternative duplication.” Both
pol3
591–598 ITD and
pol3
458–477 ITD resembled
ITDs detected in human cancer (
13
-
15
). To determine how the alternative duplication
pol3
458–477 ITD originated, we conducted WGS of WT and
rad27
Δ cells grown at 37°C or
30°C for 4 h. The mutation frequency of WT cells was the same at both temperatures (Fig.
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2A). In contrast, restrictive temperature stress increased the mutation frequency of
rad27
Δ
cells by 2-fold; in particular, the frequency of duplications and base substitutions was
increased (Fig. 2A). In addition, duplication insertions in
rad27
Δ cells grown at 37°C were
considerably longer than those in
rad27
Δ cells grown at 30°C (Fig. 2B). The duplications
revealed that
rad27
Δ cells grown at 37°C exhibited alternative duplications that were similar
to the
pol3
458–477 ITD. The alternative duplications were not detected in WT cells (30°C
or 37°C) or in
rad27
Δ cells grown at 30°C (Fig. 2C), suggesting that alternative duplications
were induced by restrictive temperature stress.
We further noted that the sequences of these alternative duplications suggested formation of
three different types of hairpin structures (Fig. 2D, fig. S4A-4D, Supplementary text S3).
This supports a model of sequential actions, including conversion of a 5’ Okazaki fragment
flap to a 3’ flap, annealing of the flap to a complementary sequence, extension of the 3’
flap, realignment, and ligation of the extended 3’ flap to produce an alternative duplication,
including
pol3
458–477 ITD. Consistent with this model, our WGS data indicated that
40% of the alternative duplications also carried base substitutions at the duplication unit.
These substitutions most likely resulted from failure to remove Pol
α
-generated errors on
the 5’ flap. To determine if the restrictive temperature induced 3’ flap formation in
rad27
Δ
cells, we developed an approach to specifically label the OH group on the 3’ flap on
genomic DNA, in which 3’ OH at the nick or at the DNA end was pre-blocked with
dideoxyribonucleotides (Fig. 2E). We detected a considerable number of 3’ flaps in
rad27
Δ
cells grown at 37°C; in contrast, we detected few flaps in
rad27
Δ cells grown at 30°C, in WT
cells grown at either temperature, or in
rad27
Δ cells carrying
pol3
458–477 ITD grown at
either temperature (Fig. 2F). Furthermore, pre-incubation of Pol
δ
with genomic DNA from
rad27
Δ cells grown at 37°C could effectively remove the 3’ flaps (Fig. 2G), suggesting that
Pol
δ
may process 3’ flaps during OFM.
To define the proposed 3’ flap-based OFM mechanism, we reconstituted the sequential
reactions of 3’ flap cleavage, DNA synthesis, and ligation of oligo-based DNA substrates
(S) with a simple 3’ flap (S2 or S3; Fig. 2H and fig. S5B) or a secondary structure-forming
3’ flap (S4 or S5; Fig. 2I) for formation of type I or type II alternative duplications. In the
presence of deoxyribonucleotide, Pol
δ
could effectively cleave 3’ flap substrates S2 and S3
and stop at the junction of the 3’ flap and DNA duplex, generating ligatable DNA nicks for
DNA Lig I (Fig. 2H and fig. S5, Supplementary text S4). However, deoxyribonucleotides
inhibited cleavage of hairpin-forming 3’ flaps, and promoted extension of the annealed 3’
flap, producing ligated extended products (Fig. 2I, Supplementary text S4); this process
resembled formation of alternative duplications. When extension of the annealed 3’ flap
could not generate ligatable nicks, only unligatable extended products were produced (fig.
S6A-6D), leading to failure of 3’ flap-based OFM. The single-stranded DNA (ssDNA)
binding protein RPA had little effect on Pol
δ
-mediated 3’ flap cleavage or subsequent nick
ligation (Fig. 2H), and it slightly enhanced formation of the ligated extended products (Fig.
2I).
Using reconstitution assays, we showed that the 3’ nuclease activities of Pol
δ
and Lig I were
sufficient to complete 3’ flap processing for OFM. Other nucleases in the nuclear extract
(NE) might also be important in processing 3’ flaps, especially the hairpin-forming 3’ flap
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(fig. S7A, S7B, Supplementary text S5). However, NE from
rad27A
Δ cells, particularly
those grown at 37°C, had reduced 3’ flap processing activity (fig. S7A, S7B, Supplementary
text S5). Because we observed no significant changes in expression of major 3’ nucleases in
yeast (fig. S8), we postulated that restrictive temperature stress could also induce molecular
changes to inhibit 3’ flap processing, allowing 3’ flaps to invade into nearby homologous
sequences, leading to alternative duplications.
We next determined how the
pol3
458–477 ITD enabled
rad27
Δ cells to overcome lethal
stress. Because the
pol3
458–477 ITD did not change Pol
δ
protein levels in
rad27
Δ cells
(fig. S9), we tested if it affected biochemical properties of Pol
δ
. We assayed the DNA
polymerase and 3’ nuclease activities of a purified recombinant protein Pol
δ
complex
containing either a WT Pol3 subunit (WT Pol
δ
) or a 458–477 ITD Pol3 subunit (hereafter
called Pol
δ
-ITD). Pol
δ
-ITD could catalyze DNA synthesis but was less processive than
WT Pol
δ
during primer extension (Fig. 3A). Similarly, Pol
δ
-ITD could effectively fill the
gap, but it was less active than WT Pol
δ
in displacing the downstream DNA oligo (Fig.
3B). In addition, Pol
δ
-ITD had relatively weak 3’ exonuclease activity on DNA duplexes,
compared to WT Pol
δ
(fig. S10). However, Pol
δ
-ITD had similar activity to WT Pol
δ
in
cleaving the 3’ flap and generating a ligatable nick (fig. S11). This activity likely allows
cells carrying the
pol3
458–477 ITD to have a similar capacity as WT cells for catalyzing
3’ flap processing for OFM. In contrast, a 3’ exonuclease-dead mutant, Pol
δ
D520E, did not
cleave the 3’ flap (fig. S11), which may explain why the Pol
δ
D520E mutation is lethal at
restrictive temperature and synthetically lethal with
rad27
Δ (
16
).
We further revealed that knock-in of
pol3
mutations significantly reduced the mutation
rate of
rad27
Δ cells, as measured by Canavanine resistance (Can
r
) (Fig. 3C) but did not
affect the mutation rate of yeast cells with WT Rad27 (fig. S12). These
pol3
mutations
nearly completely suppressed the occurrence of duplications (Fig. 3D). Consistent with the
Can
r
assay results, our WGS data confirmed that
pol3
mutations reduced the frequency
of duplications and the overall mutation frequency (Fig. 3E). Duplication mutation rate
correlates with the level of 5’ flap formation (
12
). Thus, our biochemical and genetic results
demonstrate that
pol3
ITD and other point mutations can reverse the conditional lethality
phenotype by limiting 5’ flap formation in
rad27
Δ cells.
To identify the signaling pathways that induced 3’ flap- mediated OFM and led to generation
of
pol3
ITD, we compared the transcriptomes of WT and
rad27
Δ cells grown at 37°C or
30°C. We observed that genes regulated by the checkpoint kinases Mec1, Rad53, and Dun1
were significantly up-regulated in
rad27
Δ cells, especially those grown at 37°C (Fig. 4A);
consistent with this, western blot analysis confirmed that chromatin-associated Dun1 protein
was increased in
rad27
Δ cells grown at 37°C (Fig. 4B). These results suggest activation
of the Mec1-Rad53-Dun1 axis, the major signaling pathway that is activated to counteract
genotoxic stress (
17
,
18
). We further showed that downstream targets of the upregulated
genes, including the stress response genes
HUG1, RNR2, RNR3
, and
RNR4
, and the DNA
repair gene
RAD51
, were synergistically induced by
rad27
Δ and restrictive temperature
stress (fig. S13).
RAD51
is associated with inhibition of 3’ ssDNA degradation, which at
least partially explains why degradation of 3’ flaps induced by NE from
rad27
Δ cells grown
at 37°C was markedly less than degradation induced by WT NE (fig. S7A, 7B).
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To define the role of Dun1 signaling in stress-induced mutation and generation of revertants,
we deleted the
DUN1
gene in WT and
rad27
Δ cells. We observed that knockout of
DUN1
(
dun1
)Δ in WT or
rad27
Δ cells had little effect on their survival (fig. S14), 3’ flap formation
(Fig. 4C), or mutation rate at 30°C (Fig. 4D). However,
DUN1
deletion markedly reduced
restrictive temperature stress-induced 3’ flap formation (Fig. 4C) and abolished restrictive
temperature stress-induced mutations in
rad27
Δ cells (Fig. 4D). Consistent with this,
DUN1
deletion inhibited generation of
rad27
Δ revertants (Fig. 4E, Supplementary text S6).
Furthermore, all
rad27
Δ revertants in this experiment had
pol3
mutations, predominantly
the
pol3
458–477 ITD, but none of the
rad27
Δ
dun1
Δ revertants had
pol3
mutations (Fig.
4F). These findings suggest that Dun1 activation plays an important role in the development
of restrictive temperature stress-induced mutations that can reverse the lethal phenotype of
rad27
Δ cells. Consistent with this finding, blocking activation of Chk1, a Dun1 functional
analogue, significantly inhibited spontaneous lung cancer development in FEN1 mutant
mice but not in WT mice (fig. S15, Supplementary text S7). An important function of
Dun1 activation is to induce overexpression of
HUG1, RNR2, RNR3
, and
RNR4
for
deoxyribonucleotide production. Increased deoxyribonucleotide concentrations changed the
mode of action of Pol
δ
and promoted generation of ligated extended products
in vitro
(fig.
S5, fig. S16, S17, Supplementary text S8). However, when we deleted SML1, the protein
inhibitor of ribonucleotide reductase (
19
), to increase deoxyribonucleotide production, we
did not observe increased mutation rates in
rad27
Δ cells (fig. S18), suggesting that an up-
regulation of deoxyribonucleotide alone is not sufficient to promote alternative duplications.
To demonstrate the relevance of stress-induced 3’ flap-based OFM and alternative
duplications in
rad27
Δ cells to human cancers, we used whole-exome sequencing (WES) to
analyze alternative duplications in human tumors and mutant mice modeling human FEN1
mutations. Alternative duplications, similar to those in
rad27
Δ cells grown at restrictive
temperature (i.e., 3’ flap OFM-related alternative duplications), were frequent in human B
cell acute lymphoblastic leukemia (fig. S19A-19C, Supplementary text S9). In addition,
FEN1 A159V mutation, which occurs in human lung cancers (
20
), promoted 3’ flap OFM-
related alternative duplications in mice (fig. S19D, Supplementary text S9). Therefore,
mutations in FEN1 or other OFM genes may lead to 3’ flap-based OFM, and play crucial
roles for cancer cell evolution, tumor growth, and resistance.
Our current study defines error-prone processing of RNA-DNA primers during OFM (Fig.
4G). Induction of this mechanism generates alternative duplications and base substitutions.
In WT cells, the displaced 5’ RNA-DNA flap is effectively cleaved by either Rad27 alone
or by Dna2, which first cleaves the 5’ RNA-DNA flap in the middle, leaving a shorter
5’ DNA flap for Rad27 to subsequently cleave. When Rad27 is not available, other 5’
nucleases such as Dna2 alone or Exo1 are involved in inefficient 5’ flap processing (
21
,
22
). Resolution of 5’ flaps also requires an alternative pathway that is mediated by the 3’
exonuclease activities of Pol
δ
, which removes nucleotides from the 3’ end of an upstream
Okazaki fragment, generating a gap for the unprocessed 5’ flap to re-anneal for ligation
(
16
,
23
). Restrictive temperature stress activates Dun1 signaling and stimulates
de novo
production of deoxyribonucleotides, which in turn inhibits the 3’ exonuclease activity, but
not the flap nuclease activity of Pol
δ
, and induces other DNA damage responses. These
molecular changes push OFM toward transformation of an unprocessed 5’ flap into a 3’ flap,
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either through flap equilibration (
24
) or the actions of helicases such as Sgs1 or Pif1, leading
to a secondary structure that may result in alternative duplications, including Pol
δ
-ITD, in
revertant strains. In the revertants, Pol
δ
mutations limit DNA displacement, thus suppressing
5’ flap formation or allowing more time for Dna2 or Exo1 to act on the 5’ flap and bypass
the requirement for Rad27 (Fig. 4G).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We thank Dr. Richard Kolodner for the yeast strains RDKY2672, RDKY2608, RDKY2669; Dr. Peter M.J.
Burgers for the plasmids pBL335 (GST-Pol3), pBL338 (GAL1-Pol31), pBL340 (GAL10-Pol32), and pBL341
(Pol31/Pol32); Drs. Louis Prakash and Satya Prakash for the protease-deficient yeast strain YRP654 and the
plasmids pBJ1445 (Flag-Pol3) and pBJ1524 (GST-Pol31/Pol32) to express the yeast recombinant DNA polymerase
δ
complex (Pol3, Pol31, and Pol32); Dr. Wolf-Dietrich Heyer for the anti-Dun1 antibody; and Dr. Marc S. Wold
for purified recombinant yeast RPA complex. We thank Huifang Dai, Daniela Duenas, and Martin E. Budd for
technical assistance in mouse and yeast genetic experiments and stimulating discussions. We thank Drs. Keely
Walker and Sarah Wilkinson for critical reading and editing of the manuscript.
Funding:
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 National Institutes of Health under award number P30 CA033572.
Data and materials availability:
All data is available in the manuscript or the supplementary materials. Accession numbers
for mouse and yeast genomics datasets are GSE181154 and GSE178876, respectively.
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Fig. 1. Pol
δ
internal tandem duplication (ITD) and missense mutations drive resistance to
rad27
Δ-induced conditional lethality.
(A)
Spot assays of WT,
rad27
Δ, or
rad27
Δ revertant (Rev) yeast cells grown at 30°C
(optimal temperature), 25°C (sub-optimal temperature), or 37°C (restrictive temperature)
for 48 h.
rad27
Δ::URA3 and
rad27
Δ::LEU2 represent the
rad27
Δ allele with a linked
URA3 or LEU2 selection marker gene, respectively.
(B)
pol3
mutations detected in
independent revertant strains (
n
=31). Circles and diamonds represent base substitution and
ITD mutations, respectively. The domain structures were defined as previously described
(
23
).
(C)
Spot assays of WT,
rad27
Δ, or
rad27
Δ yeast cells with indicated
pol3
knock-in
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mutations grown at 30°C, 25°C, or 37°C for 48 h. POL3::HIS3 represents the POL3 (WT or
mutant) alleles with a linked HIS3 selection marker gene.
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Fig. 2. Restrictive temperature stress induces 3’ flap-based OFM and results in alternative
duplications.
(A)
Somatic mutation frequencies and types as determined by WGS, in WT and
rad27
Δ
cells grown at 30°C or 37°C for 4 h.
(B)
Lengths of inserted DNA sequences in
duplications in
rad27
Δ cells grown at 30°C or 37°C for 4 h.
(C)
Top, diagram of classic
and alternative duplications. Bottom, frequencies of classic and alternative duplications.
(D)
Predicted structures leading to three types of alternative duplications. Red and green
lines: DNA sequences in red and green in fig. S4A; Orange lines: yellow highlighting in
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