Science - USA (2021-12-03)

1256 3 DECEMBER 2021¥VOL 374 ISSUE 6572 science.orgSCIENCE

G

0

20

40

60

80

100

120

rad27Δ

rad27Δ

dun1Δ

0

50

100

150

200

250

300

Mutation Rate (

×10

-^7
)

WT

rad27Δ

rad27Δ

dun1Δ

dun1Δ

0 0.2 0.4 0.6 0.8 1

DUN1 RAD53

MEC1 TEL1

0.2 0

rad27Δ30°C vs

WT 30°C

WT 37°C vs

WT 30°C

rad27Δ37°C vs

rad27Δ30°C

5.2 × 10 -5

7.3 × 10 -12

p values

1.4 × 10 -13

2.3 × 10 -26

0.00032

1.8× 10 -8

1.0 × 10 -6

n.s.

0.00016

2.8 × 10 -18

9.3 × 10 -14

n.s.

n.s.

p values

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

0.015

Up-regulated gene ratio

Pathway:

Down-regulated gene ratio

A

0

20

40

60

80

100

120

POL3

mutated revertant (%)

0

rad27Δrad27Δ

dun1Δ

F

WT rad27Δ dun1Δ

30 37 30 37 30 Temp. (°C)

α-Dun1

α-Histone H2B

B

Normal (30°C) Restrictive (37°C)

D

n.s.

p = 0.02

Incubation time (h) at 37°C

Revertant frequency (

×^10

-4)

E

0 2 4 8 24

C

dun1

Δ

rad27

Δ

dun1

Δ

rad27

Δ

dun1

Δ

rad27

Δ

rad27

Δ

dun1

Δ

30°C 37°C

WT WT

Total DNA input

32 P- 3’ flap

0

0.5

1

1.5

2

2.5

Relative Dun1 level

p < 0.001

p < 0.001

p = 0.056

30 37 30 37(°C)

WT rad27Δ

WT rad27Δ mutant rad27Δ revertant

(Polδ ITD)

Fig. 4. Restrictive temperature stress activates signaling that facilitates error-
prone OFM and generation ofrad27Drevertants.(A) Up-regulated or down-
regulated gene ratios in Dun1-, Rad53-, Mec1-, or Tel1-controlled pathways in WT or
rad27Dyeast cells exposed to 30°C (4 hours) or 37°C (4 hours).pvalues were
calculated using the hypergeometric test; n.s. indicates not significant. (B)A
Western blot of chromatin-associated Dun1 protein in WT orrad27Dcells exposed to
30°C (4 hours) or 37°C (4 hours) is shown at the top. Histone H2B was used as a
loading control for the chromatin fraction in each sample.dun1Dis a negative
control. Quantification of chromatin-associated Dun1 relative to the loading control is
shown at the bottom. The Dun1 level inrad27Dcells grown at 30°C was arbitrarily
set as 1, and the relative Dun1 levels in other samples were calculated by dividing
their Dun1 levels by that inrad27Dcells grown at 30°C. Error bars indicate SEM
(n= 4 biological replicates). (C) Levels of 3′flaps in genomic DNA from WT,dun1D,

rad27D, orrad27Ddun1Ddouble-mutant cells grown at 30° or 37°C for 4 hours.

(D) Mean Canrmutation rates of WT,rad27D,dun1D, andrad27Ddun1Dcells exposed

to 30°C (4 hours) or 37°C (4 hours). Error bars indicate SD (n= 3 independent

assays). (E) Median revertant frequencies ofrad27Dorrad27Ddun1Dcells (n=

3 independent assays). (F) Percentage ofrad27Dorrad27Ddun1Drevertants that

carry apol3mutation (n= 19 for each strain). (G) Schematic illustrating error-free

5 ′flap–mediated OFM in WT cells, error-prone 3′flap–mediated OFM and the

corresponding consequences inrad27Dcells under restrictive temperature stress

(37°C), and the impact of Pold-ITD on OFM in the revertant. Blue and pink segments

represent primase- and Pola-synthesized RNA primer and DNA. The red segment

represents Pold-synthesized DNA, which replaces Pola-synthesized DNA. The

green segment represents Pold-synthesized spacer DNA as part of alternative

duplication. Black dots represent Polaincorporation errors.

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