research summary of DNA replication and repair, kidney diseases.

1. Summary of research and ideas

2. Reprogramming cancer cells
Hendrix, M., Seftor, E., Seftor, R. et al. Gong, L., Yan, Q., Zhang, Y. et al.

3. Bo Song
Mouse ES cell OS25
2 weeks

4. PKA-dependent hyperactivation of hepatic IRE1α contributes to obesity-associated
disruption of glucose metabolism
Mao, Ting, et al. "PKA phosphorylation couples hepatic inositol-requiring enzyme 1α to
glucagon signaling in glucose metabolism." Proceedings of the National Academy of
Sciences 108.38 (2011): 15852-15857.

6. Bubeck, et al. (2011)
3’ 5’
Flap endonuclease 1 (FEN1) removes 5’ ssDNA flaps from DNA
3’ 5’

7. FEN1 crystal structures show conformational changes of DNA
Tsutakawa, et al.(2017)
FEN1D86N- Substrate
FEN1D233N-Product
Close-up view
(5K97)
FEN1D86N-
Substrate
Close-up view
(5UM9)
• Bending of DNA substrate by 100°;
• Increase of interstrand and decrease of intrastrand
base stacking;
• Change of stacking pattern between Y40 and +1/-1
nucleotide.
A
A
B
K+

8. Aims of the project
• To measure rate of individual steps in the reaction;
• To find the order of events and rate limiting steps to resolve the
mechanism.

9. 0.03
0
0.005
0.008
0.01
0.015
0.02
0.025
0.04
Time
(s)
hFEN1 cleaves DNA 5’-flap
0.06
0.075
0.08
0.09
0.18
0.2
0.3
0.4
S (33 nt)
P (15 nt)
kcleavage = 24 ± 0.8 s-1
• kcleavage = 24 ± 0.8 s-1
• kcat = 1.5 ± 0.004 s-1
STO condition: [DNA]= 0.25 μM, [FEN1]= 2.5 μM.
Steady-state condition: [DNA]= 0.8 μM, [FEN1]= 2 nM.
Buffer: 30 mM Hepes-KOH (pH 7.4), 40 mM KCl, 8 mM MgCl2.

10. Förster resonance energy transfer (FRET) fundamentals
Stejskalova, Eva & Stanek, David. (2012). International journal
of molecular sciences. 13. 14929-45. 10.3390/ijms131114929.

11. hFEN1 bends DNA at a diffusion-limited rate
• The first phase is dependent on [FEN1] and reflects DNA binding & bending
• The second phase likely reports fast unbending of nicked product after flap cleavage
9

12. DNA distortion limits the cleavage rate
DNA distortion? Flap cleavage
WT
kdistortion = 21 ± 0.2 s-1
WT
kcleavage = 24 ± 0.8 s-1
The 2AP signal change reflects DNA distortion at flap junction?

13. The D34A mutant may uncouple cleavage and DNA distortion
Red sphere: Metal ion B; Yellow sphere: ion A
D34
+1NT
-1NT
α2 α4
P
A
B
8Å

14. kdistortion = 21±0.2 s-1
DNA distortion limits the cleavage rate
The 2AP signal change reflects DNA distortion at flap junction.
kdistortion = 0.1±0.004 s-1
D34A
WT
2AP assay
WT
D34A
WT
kcleavage = 24±0.8 s-1
Flap cleavage

15. FEN1DNAopen
kon
3 ×109 M-1 s-1
A minimal kinetic scheme of FEN1
FEN1+ DNA FEN1DNA FEN1DNAbent
kbend
FEN1+DNAnick
FEN1DNAnick
kdistortion krelease
kcleavage
fast ~24 s-1 fast 1.5 s-1
A FRET assay revealed that hFEN1 binds and bends a 5’ flap DNA
substrate rapidly at a diffusion-limited rate (109 M-1s-1).
A 2-aminopurine fluorescence-based assay revealed distortion of DNA at
the flap junction at a much slower rate of ~24 s-1.
Quench-flow measurements revealed that this step limits the cleavage rate
to ~24 s-1.
A subsequent product release limits the overall steady state turnover rate to
~1.5 s-1.
Conserved active site residues contribute to an electrostatic environment
that stimulates cleavage of the 5' flap.
5’flap

16. ZBP1 recognizes Z-nucleic-acid to induce inflammation and cell death

17. IFNγ (10 ng/ml)
LPS ( 5 ug/ml) : - + - + - + - + - + - +
ZBP1 mediates NLRP3, Casp1 and RIPK1 activation induced
by IFN γ& LPS in TCMK-1
GAPDH
EV 1 sgZBP1 1 EV 2 sgZBP1 2
P-RIPK1
Casp1
ZBP1
EV 3 sgZBP1 3
75
50
75
100
50
kD
37
24hrs
NLRP3
RIPK1

18. IFNγ (10 ng/ml)
LPS ( 5 ug/ml) : - + - + - + - + - + - +
ZBP1 mediates pyroptosis and necroptosis induced by IFN γ&LPS in TCMK-1
GAPDH
EV 1 sgZBP1 1 EV 2 sgZBP1 2
P-MLKL
GSDMD
Cleaved
GSDMD
N-terminal
C-terminal
ZBP1
EV 3 sgZBP1 3
50
37
20
MLKL 50
50
50
kD
37
24hrs
EV= Empty vector

19. IFNγ (10 ng/ml)
LPS ( 5ug/ml) : - + - + - + - + - + - +
EV 1 sgZBP1 1 EV 2 sgZBP1 2 EV 3 sgZBP1 3
20
37
15
37
kD
24hrs
ZBP1 mediates inflammation, pyroptosis and apoptosis induced
by IFN γ&LPS in TCMK-1
Pro-Casp3
Cut Casp3
IL-1β
Cut IL-1β
P-p65
50
GAPDH
37

20. 10ng/ml IFN γ in each LPS treatment group
ZBP1 knockdown protects tubular cell line TCMK-1 against
cell death induced by IFN γ /LPS
EV: + - + - + - + -
sgZBP1: - + - + - + - +
LPS (μg/ml): 0 0 0.1 0.1 1 1 10 10
EV: + - + - + - + -
sgZBP1: - + - + - + - +
LPS (μg/ml): 0 0 0.1 0.1 1 1 10 10

21. IFNγ (10 ng/ml)
LPS ( ug/ml) : 0, 0, 0.5, 0.5, 5, 5, 0, 0, 0.5, 0.5, 5, 5
ZBP1 KO does not reduce inflammation induced by IFN γ & LPS in BMDM
WT KO WT KO WT KO WT KO WT KO WT KO
ZBP1
3 hrs
IL-1B
GAPDH
NLRP3
50
kD
100
150
37
37

22. ZBP1 KO does not reduce LPS-induced AKI in mice
Casp-3
Pro-Casp1
GAPDH
10mg/kg LPS
24hrs
LPS: - + - +
WT KO
Cut Casp1
NGAL
*
NS
kD
50
WT ZBP1 KO
25
37
25
37

23. TGF-β1: - + - + - +
ZBP1 regulates fibrosis markers in fibroblast 3T3 cells
EV sgZBP1 a sgZBP1 b
GAPDH
ZBP1
α-SMA
FN
2ng/ml
24 hours treatment
260
kD
50
50
37

24. ZBP1 KO does not reduce UUO induced kidney fibrosis in mice
Col-I
GAPDH
WT ZBP1 KO
Con1 UUO1 Con1 UUO1
a-SMA
100
50
10 days UUO
FN 260
37
kD
WT Con
WT UUO
KO UUO
KO Con
WT Con
WT UUO
KO UUO
KO Con
WT Con
WT UUO
KO UUO
KO Con
WT ZBP1 KO WT ZBP1 KO
WT ZBP1 KO

25. WT ZBP1 KO
NS
ZBP1 KO does not reduce UUO induced kidney fibrosis in mice
CON UUO
WT
ZBP1 KO
Sirius Red Stain

26. The hypothesis: exosomal mtNAs originated from damaged kidney epithelial
cell trigger cGAS in macrophages to initiate inflammatory responses
TEC
Damage
e.g.
AngII,H2O2
Exosome
cGAS
mtNA
mtNA
PANoptosis
Macrophage
Endocytosis
Kidney injury

27. Exosomes isolated from TCMK-1 cells

28. Exosomes isolated from TCMK-1 cells after AngII treatment
10 uM Ang II 24 hrs

29. Uptake of Exosome by BMDM
Control 200ul Exosome-PKH67 400ul Exosome-PKH67
DAPI
PKH67
Exosomes were isolated from AngII treated TCMK-1 cells.
After labeling with PKH67, exosomes were incubated with
BMDM for 4 hours.

30. The inhibition of exosome uptake by an endocytosis inhibitor
Control 100ul Exosome-PKH67 220ul Exosome-PKH67
DAPI
PKH67
Exosomes were isolated from AngIItreated TCMK-1 cells.
After labeling with PKH67, exosomes were incubated with
RAW cells for 3 hours.
Dynasore 80 ug/ml
400 X
Inhibitor
No
inhibitor

31. Knockdown of Rab27a inhibits inflammation in BMDM activated by TCMK1
conditional media
IL-1β/GAPDH
mRNA
(Fold
change)
MCP-1/GAPDH
mRNA
(Fold
change)

32. AngII treated TCMK-1 cells release exosomes containing DNA
1Kb plus ladder
From Ang II treated cell
Control
DNA (ug): 0.1 0.2 0.8 0.1 0.2 0.8
Exosome DNA after Pheno-Chloroform extraction
1.5kb
2% agarose gel

33. Ang II promotes mtDNA level in exosomes released by TCMK1
Relative
expression
(ND1/GAPDH)
(fold
change)
***