Science - USA (2021-12-03)

inrad27Dcells, 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 preblocked with di-
deoxyribonucleotides (Fig. 2E). We detected a
considerable number of 3′flaps inrad27Dcells

grown at 37°C; by contrast, we detected few

flaps inrad27Dcells grown at 30°C, in WT cells

grown at either temperature, or inrad27Dcells

carryingpol3 458 – 477 ITD grown at either tem-

perature (Fig. 2F). Furthermore, preincubation

of Poldwith genomic DNA fromrad27Dcells

grown at 37°C could effectively remove the 3′

flaps (Fig. 2G), suggesting that Poldmay pro-

cess 3′flaps during OFM.

To define the proposed 3′flap–based OFM

mechanism, we reconstituted the sequential

reactions of 3′flap cleavage, DNA synthesis,

SCIENCEscience.org 3 DECEMBER 2021•VOL 374 ISSUE 6572 1255

M

• WT ITD

60

nt

30

25

20

50

40

100

80

A

Primer

extension

products

Substrates

Substrates

Gap filling

products

Time

• WT ITD Polδ
  M

60

nt

30

25

20

50

40

100

80

Displacement DNA

synthesis products

B

Time

Polδ

Mutations per million bases

WT

rad27Δ

POL 3

0

20

40

60

80

100

Duplication

Base Substitution

Frame-shift

Other

Mutation spectrum (%)

WT

rad27Δ

POL3

DE

0

10

20

30

40

50

60

70

(

Mutation Rate ×10

-7)

WT

rad27Δ

POL3

0.0001

p values

0.0009

0.0006

0.0003

0.0009

C

0

1

2

3

4

5

6

7

8

Duplication

Base Substitution

Di-, tri-nucleotide repeat indel

Frame-shift

Other

(^32) P
3’
25nt 150nt
5’
3’
5’
3’
3’ 180nt 180nt

• (^32) P


• 25nt
  Fig. 3. Pold-ITD suppresses 5′flap formation.(AandB) In vitro assays of
  primer extension (A) and displacement DNA synthesis (B) by WT Poldor
  Pold-ITD. DNA substrates and primer extension products in (A) and DNA
  substrates, gap-filling products, or displacement DNA synthesis products in
  (B) are indicated. (C) Mean Canrmutation rates of WT (n= 5),rad27D(n= 5), or
  rad27Dyeast cells with knock-in ofpol3 458 – 477 ITD (n= 3),pol3R470G
  (n= 2),pol3R475I (n= 3),pol3A484V (n= 2), andpol3S847Y (n= 2). Error
  bars indicate SD;pvalues were calculated using Student’sttest. (D) Canr
  mutation spectra of the indicated yeast strains. Values shown are percentages of
  the specific type of Canrmutation in WT (n= 22),rad27D(n= 20), orrad27D
  with knock-in ofpol3 458 – 477 ITD (n= 21),pol3R470G (n= 10),pol3R475I
  (n= 21),pol3A484V (n= 19), orpol3S847Y (n= 21). (E) Mutation frequencies
  and types present across the genome, as determined by WGS, in WT,rad27D,
  orrad27Dcells with indicatedpol3knock-in mutations grown at 30°C (n= 1).
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