1254 3 DECEMBER 2021¥VOL 374 ISSUE 6572 science.orgSCIENCE
S4M++
+
+++
+
+
+++
++S3Cleaved
S3nt5025403010080601502015Ligated
P3S3A051015(^20) Duplication
Base substitution
Frame-shift
Di, tri-nucleotide repeat indel
Other (complex)
Mutations per million bases
WT rad2 7 Δ WT rad27Δ
30°C 37°C(Stress)
5’
3’
3’
5’
5’
3’
Type 1:
3’ flap invasion
5’
3’
3’
5’
3’
Type 3:
3’ flap fold-back and invasion
5’ 5’
3’
3’
5’
3’
Type 2:
3’ flap fold-back
5’
E
D
fragmentation
blocking nick, end 3’ OH
(^32) P labeling flap 3’ OH
0
5
10
15
30°C 37°C
Duplication mutation length (nt)
Duplication mutation counts
B
Duplications per
million bases
WTrad27Δ
C
0
2
4
6
Classic
Alternative
WTrad27Δ
30°C 37°C
H
Ligated
P4, P5
S5
Extended
S4, S5
Polδ
RPA
dNTP
Lig I
++
+
+++
+
+
+S5
++
++150nt60305040100
8025
20++
+
+++
+
+
+S4
++
++
M +
ICleaved S4Polδ
RPA
dNTP
Lig IG-+Polδrad27Δ
3 7° C
rad27Δrad27ΔWT30°C 37°CWTTotal DNA input32P- 3 ’ f l a pFrad27ΔITD 30°Crad27ΔITD 37°CFig. 2. Restrictive temperature stress induces 3′flapÐbased OFM and results
in alternative duplications.(A) Somatic mutation frequencies and types, as
determined by WGS, in WT andrad27Dcells grown at 30° or 37°C for 4 hours.
(B) Lengths of inserted DNA sequences in duplications inrad27Dcells grown at
30° or 37°C for 4 hours. The dashed lines indicate the trend lines of corresponding
bar graphs. nt, nucleotide. (C) A diagram of classic and alternative duplications
is shown at the top. Frequencies of classic and alternative duplications are shown
at the bottom. (D) Predicted structures leading to three types of alternative
duplications. Red and green lines represent the DNA sequences in red and green in
fig. S4A; orange lines represent the orange-highlighted DNA sequences in fig. S4,
AtoD.(E) Schematic for specific labeling of 3′flaps in genomic DNA. Green dots
indicate dideoxyribonucleotide; the red star indicates^32 P-deoxyribonucleotide.
(F)Levelsof3′flaps in genomic DNA from WT,rad27D, orrad27Dpol3ITD
(rad27D-ITD) cells grown at 30° or 37°C for 4 hours. (G)Levelsof3′flaps in
genomic DNA fromrad27Dcells grown at 37°C with or without pretreatment
with Pold.(HandI) Reconstitution assays using^32 P-labeled 3′flap substrate S3
(H) or^32 P-labeled secondary structure–forming 3′flap substrate S4 or S5 (I).
Substrate structures are shown above a representative image of 8% denaturing
polyacrylamide gel electrophoresis (PAGE). DNA substrates (S3, S4, S5),
cleavage products (cleaved S3, S4), unligated extended 3′flap intermediates
(extended S4, S5), and ligated extended products (ligated P3, P4, P5) are
indicated. dNTP, deoxyribonucleotides; M, marker.RESEARCH | REPORTS