Supplementary Legends
Figure S1. Schematics of oligonucleotide substrates designed for the RNA primer
removal (RPR), Pol α error editing (AEE) and nuclease activity assays.
A. Set 1 (A1 - 4). Gapped flap substrates with an “A” in the first position of a three or fournucleotide (nt) single-stranded (ss) DNA region and with or without a “C/T” mismatch
in the downstream duplex were designed for the OFM and AEE assays. With the
mismatched substrates, the C is unique in the downstream duplex region. In this way,
when a radio-labeled nt T or G is added, we will be able to monitor the reactions for flap
removal, gap filling and ligation (OFM) or AEE. The second pair, with a unique T in the
ss DNA region and a G in the downstream duplex, was designed using the same
principle, gave similar results, and is used interchangeably with the first pair.
B. Set 2 (B1 - 4). The substrates contain a variable distance (in nt) between the first paired
nt of the downstream duplex and mismatched nts (indicated as X nt), where X nt is 3, 6,
12, or 18 nts.
C. Set 3 (C1 - 4). These substrates were designed to test the nuclease activity of FEN1
wild-type (WT) and mutant enzymes. They include substrates forming a duplex DNA
either containing double flaps (a 3’ single nt flap and a 40 nt long 5’ flap), without a 3’
flap but with different lengths of 5’ flaps (long 5’ flap = 40 nt and short 5’ flap = 5 nt), or
containing the nicked duplex DNA without any flap.
D. Set4 (D1 - 4). These substrates contain gaps or nicks with or without an upstream 3’
flap.
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E. Set5 (E1, E2). These two substrates are similar to A1/A2; the only difference is that
E1/E2 contain RNA flaps in place of the DNA flaps in A1/A2.
Figure S2. RNA primer removal (RPR) and α-segment error editing (AEE) assays
using RNA flap-containing substrates.
A. RPR assays with RNA flap-containing substrates with or without a mismatch in the
downstream duplex DNA region (E1 or E2) using nuclear extracts (NEs). 1 μg of NE was
incubated with 500 fmol of the gap substrates containing a 15 nt RNA flap at 37 °C for 5,
10, 20, 40, 60, and 80 minutes (lanes 3-8 for E1 or 9-14 for E2). Lane 2 is the reaction
with E1 but without adding any NE. All reactions were carried out in a buffer containing
5 μCi [α-32P] dTTP and 50 μM dGTP, dCTP, and dATP.
B. AEE assays with RNA flap-containing substrates without (E1) or with (E2) a mismatch
in the downstream duplex DNA region. 1 μg of NE was incubated with 500 fmol of the
gap substrates containing a 15 nt RNA flap at 37 °C for 5, 10, 20, 40, 60, and 80 minutes
(lanes 3-8 for E1 or 9-14 for E2). Lane 2 is the reaction with E1 but without adding any
NE. All reactions were carried out in a buffer containing 5 μCi [α-32P] dGTP and 50 μM
dATP, dCTP, and dTTP.
Figure S3. Protein expression and purification.
A. His-tagged FEN1 and mutant proteins were expressed in E. coli cells and purified based
on a protocol that we published previously(Liu et al, 2006). Lane 1 shows the molecular
weight markers and lane 2 shows the purified FEN1 protein.
B. DNA2 protein was expressed in human 293T cells and purified based on a protocol that
we published recently (Ronchi et al, 2013). Lane 1 shows the molecular weight markers
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and lane 2 shows the purified DNA2 protein. C. EXO1 protein was expressed in insect
cells and purified based a protocol that we have published previously (Zhang et al,
2005). Lane 1 shows the molecular weight markers and lane 2 shows the purified EXO1
protein.
Figure S4. Nuclease activity profiles of FEN1, EXO1, and DNA2.
A. Substrates with a double-flap DNA duplex containing a 3’ single nt flap and a 40 nt long
5’ flap, a single 40 nt long 5’ flap, a single 5 nt short 5’ flap, and a nick structure were 32P
labeled at the 5’ flap or 5’ end of the downstream oligonucleotide. 500 fmol of FEN1,
EXO1 or DNA2 was incubated with the substrate at 37 °C for 60 min. The reaction
products were collected and run on a denatured PAGE gel and were visualized by
autoradiography. Lane 1: molecular weight markers (nt); lanes 2, 6, 10, and 14:
substrates only; lanes 3, 7, 11, and 15: substrates incubated with FEN1; lanes 4, 8, 12,
and 16: substrates incubated with DNA2; lanes 5, 9, 13, and 17: substrates incubated
with EXO 1.
Figure S5. Nuclease activity profiles of FEN1 and its mutant variants E160D and
A159V.
A. and E. The cleavage efficiency of mFEN1, mE160D and mA159V for the double flap
substrate. 500 fmol of mFEN1, mE160D or mA159V was incubated with a double-flap
DNA duplex with a 3’ single nt flap and a 40 nt long 5’ flap.
B. and F. The cleavage efficiency of mFEN1, mE160D or mA159V with the single long 5’
flap substrate. 500 fmol of mFEN1, mE160D or mA159V incubated with a 40 nt long 5’
flap substrate.
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C. and G. The cleavage efficiency of mFEN1, mE160D or mA159V with the single short 5’
flap substrate. 500 fmol of mFEN1, mE160D or mA159V incubated with a 5 nt long 5’
flap substrate.
D. and H. The cleavage efficiency of mFEN1, mE160D or mA159V with the nick substrate.
500 fmol of mFEN1, mE160D or mA159V incubated with a 5’ downstream labeled nick
substrate. All of the reactions were carried out at 37 °C for up to 60 min. The reaction
products were taken from different time points, as indicated, and run on a denatured
PAGE sequencing gel. Products were visualized by autoradiography.
Figure S6. Knock-in of A159V FEN1
A. The expected restriction maps of the A159V targeting vector and different types of Fen1
alleles. A DNA fragment containing the mouse C476T Fen1 mutation (A159V in amino
acid sequence) was generated by site-directed mutagenesis. It was then ligated into the
gene targeting vector, PKO scrambler NTK (Invitrogen). The remaining copy of the Lox
P sequence is located in the intron sequence, 200 bp upstream of the ATG start codon of
the Fen1 open reading frame.
B. Southern hybridization of WT and A159V (neo+) heterozygous (AV +/-) embryonic
stem (ES) cells. Genomic DNA isolated from ES cells was digested with Hind III or Xho I
and detected with probes 1 and 2, respectively.
C. Selection of mice heterozygous for A159V. Fen1 alleles were amplified by PCR using tail
genomic DNA as a template.
Figure S7. Deficiency of MSH2 or MLH1 causes mismatch repair (MMR) impairment
but only MSH2 and not MLH1 leads to AEE impairment in the in vitro reactions.
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A. In vitro assay to determine MMR efficiency in wild-type (HeLa), or MSH2- or MLH1deficient cell lines. NALM-6 (lymphoblast) and HCT116 (colorectal) cell lines are MSH2deficient and MLH1-deficient, respectively. Heat-inactivated (HI) extract derived from
MMR-proficient HeLa cells was used as a negative control. The left panel in panel A
shows how the MMR assay works. The right panel shows MMR activity of each indicated
cell line using Southern blotting analysis.
B. MSH2 deficiency but not MLH1 deficiency impairs FEN1-mediated AEE. TK-6 is an
MMR-proficient lymphoblast cell line, and was used as a positive control. Incubation
times are 5, 10, 20, 40, and 60 minutes at 37°C.
Figure S8. Transformation frequency of mouse FEN1 A157V knock-in cells.
A. Image of transformed cell colonies. Mouse embryonic fibroblast (MEF) cells were
trypsinized and seeded onto a new dish (105 cells per dish) for the colony-focus
formation assays. The cells were incubated at 37 °C for 30 days before fixing with cold
methanol and staining with Giemsa.
B. Transformation frequency of WT and WT/A159V MEF cells. The error bars were
generated based on three independent experiments.
Figure S9. Cancer frequency of FEN1 A157V knock-in mice.
A. H & E staining of normal mouse lung.
B. H & E staining indicating the presence of bronchiole-alveolar adenoma in the A159V
FEN1 mutant mouse.
C. H &E staining of normal mouse spleen.
D. H & E staining indicating the presence of lymphoma in the A159V FEN1 mutant mouse.
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E. H & E staining of normal mouse skin.
F. H & E staining indicating the presence of squamous cell carcinoma in the A159V FEN1
mutant mouse.
G. Tumor frequency of WT and WT/A159V mice in various organs.
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