Autocyclized and oxidized forms of SCR7 induce cancer cell death by inhibiting nonhomologous DNA end joining in a Ligase IV dependent manner

Article abstract of DOI:10.1111/febs.14661

Nonhomologous DNA end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals. Previously, we have described a small molecule inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV-dependent manner. Administration of SCR7 within the cells resulted in the accumulation of DNA breaks, cell death, and inhibition of tumor growth in mice. In the present study, we report that parental SCR7, which is unstable, can be autocyclized into a stable form. Both parental SCR7 and cyclized SCR7 possess the same molecular weight (334.09) and molecular formula (C₁₈H₁₄N₄OS), whereas its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C₁₈H₁₂N₄OS), molecular weight (332.07), and structure. While cyclized form of SCR7 showed robust inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was minimal on Ligase III, Ligase I, and T4 DNA Ligase-mediated joining. Importantly, both forms inhibited V(D)J recombination, although the effect was more pronounced for SCR7-cyclized. Both forms blocked NHEJ in a Ligase IV-dependent manner leading to the accumulation of DSBs within the cells. Although cytotoxicity due to SCR7-cyclized was Ligase IV specific, the pyrazine form exhibited nonspecific cytotoxicity at higher concentrations in Ligase IV-null cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV-dependent manner, although SCR7-pyrazine is less specific to Ligase IV inside the cell.

Full text of DOI:10.1111/febs.14661

Autocyclized and Oxidized Forms of SCR7 Induce Cancer Cell
Death by Inhibiting Nonhomologous DNA End joining in a Ligase IV
Dependent Manner
1
2*
3,4*
1*
Supriya V. Vartak , Swarup H. Ashok , Vidya Gopalakrishnan , Vindya K.G. , Virginie
5
1
6
2
1
Ropars , Mridula Nambiar , Franklin John , Kothanahally S. Sharath Kumar , Rupa Kumari ,
Nitu Kumari , Ujjayinee Ray , Gudapureddy Radha , Depina Dinesh , Monica Pandey ,
Hanumappa Ananda , Subhas S. Karki , Mrinal Srivastava , Jean Baptiste Charbonnier ,
1
1
1
1
1
1, 2
7
1
5
3**
2**
1**
Bibha Choudhary , Mantelingu Kempegowda and Sathees C. Raghavan
1
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India;
2
3
Department of Chemistry, University of Mysore, Mysuru, India; Institute of Bioinformatics
and Applied Biotechnology, Electronics City, Bangalore 560 100, India; Manipal Academy of
4
5
Higher Education, Manipal, Karnataka, India; Institute for Integrative Biology of the Cell
(
I2BC), CEA, CNRS, Univ Paris-Sud, Univ Paris-Saclay, 91198 Gif-sur-Yvette cedex,
6
7
France, Department of Chemistry, Sacred Heart College, Kochi 682 013, India; Department
of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Bangalore 560 010,
India.
*
Equal second authors
*Corresponding authors
*
Bibha Choudhary
Ph. +91 80 2852 8900 extn 105; Fax:+91 80 2852 8904
e-mail : vibha@ibab.ac.in
Mantelingu Kempegowda
Sathees C. Raghavan
Ph. +91 82 1241 9497; Fax: +91 82 1250 0846
e-mail : kmlingu@gmail.com
Ph. +91 80 2293 2674; Fax: +91 80 2360 0814
e-mail : sathees@iisc.ac.in
Running title: Cyclized and oxidized forms of SCR7 inhibit NHEJ
Received Date : 15-Jun-2018
Revised Date : 23-Aug-2018
Accepted Date : 17-Sep-2018
Article type : Original Articles
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/febs.14661
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Key words: DNA Ligase, Inhibitor of DNA repair, V(D)J recombination, Double-strand break,
NHEJ, End-joining, Homologous recombination, CRISPR, Genome editing
Abbreviations
NHEJ, Nonhomologous End Joining; DSB, Double-strand Break; IR, Ionizing Radiation;
MMEJ, Microhomology Mediated End Joining; HR, Homologous Recombination; ITC,
Isothermal Titration Calorimetry; NMR, Nuclear Magnetic Resonance; DMF,
Dimethylformamide; DMSO, Dimethyl sulfoxide; V(D)J, Variable Diversity Joining; MRN
complex, MRE11, RAD50 and NBS1; IC50, Inhibitory Concentration 50; HDR, Homology
Directed Repair; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; Cas,
CRISPR associated; TLC, Thin Layer Chromatography; PAGE, Polyacrylamide Gel
Electrophoresis, A, Ampicillin; CA, Chloramphenicol-Ampicillin; PSLU, Photostimulated
Luminescence Unit; L4/X4, Ligase IV/XRCC4
ABSTRACT
Nonhomologous DNA end joining (NHEJ) is the major DNA double-strand break
(
DSB) repair pathway in mammals. Previously, we have described a small molecule
inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV-dependent manner. Administration of
SCR7 within the cells resulted in the accumulation of DNA breaks, cell death and inhibition
of tumor growth in mice. In the present study, we report that parental SCR7, which is
unstable can get autocyclized into a stable form. Both parental SCR7 and cyclized SCR7
possess the same molecular weight (334.09) and molecular formula (C H N OS), whereas
18
14
4
its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C H N OS),
18
12
4
molecular weight (332.07) and structure. While cyclized form of SCR7 showed robust
inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized
forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was
minimal on Ligase III, Ligase I and T4 DNA Ligase-mediated joining. Importantly, both forms
inhibited V(D)J recombination, although the effect was more pronounced for SCR7-cyclized.
Both forms blocked NHEJ in a Ligase IV-dependent manner leading to accumulation of
DSBs within the cells. Although cytotoxicity due to SCR7-cyclized was Ligase IV-specific, the
pyrazine form exhibited non-specific cytotoxicity at higher concentrations in Ligase IV-null
cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we
report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV-dependent
manner, although SCR7-pyrazine is less specific to Ligase IV inside the cell.
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INTRODUCTION
Failure to repair DNA double-strand breaks (DSBs) could lead to cell death, while
their misrepair may result in chromosomal abnormalities and oncogenesis [1-5].
Homologous recombination (HR) and nonhomologous DNA end joining (NHEJ) are two
major DSB repair pathways that efficiently repair DSBs in mammals in a cell cycle phase
dependent manner [2, 4, 6-9]. Both pathways are well characterized at the mechanistic level
and involve different enzymatic machinery. In addition to the classical NHEJ, an alternate
NHEJ pathway has been described, that can operate in a microhomology-dependent
manner, called microhomology mediated DNA end joining (MMEJ), which utilizes an entirely
different set of proteins during DSB joining [6, 10-13].
Among DSB repair pathways, NHEJ corrects majority of the DSBs generated within a
cell, as it operates throughout the cell cycle. During NHEJ, KU70/80 proteins bind to the
DNA ends and recruit other DNA modifying enzymes such as DNA-PKcs, Artemis and
polymerases to DNA ends. DNA Ligase IV/XRCC4 along with XLF and PAXX help in final
ligation of DNA ends [2, 4, 6, 7, 14]. In case of MMEJ, Ligase III and/or Ligase I along with
other proteins help in repair of DSBs, whereas during HR, the Ligase involved is Ligase I [6,
8-10, 12, 15, 16].
Previously, we have described a novel approach to induce cytotoxicity in cancer cells
by inhibiting NHEJ using a small molecule inhibitor, SCR7 [17]. SCR7 blocked NHEJ in a
Ligase IV dependent manner both in vitro and ex vivo leading to accumulation of DNA
double-strand breaks (DSBs) within the cells, activating apoptosis. Among the four mouse
tumor models studied, three showed significant tumor regression upon treatment with SCR7
[
17]. The use of SCR7 as a biochemical inhibitor was also reported for studying the
organization and dynamics of NHEJ proteins during a double-strand break and studies
involving chromosomal territory relocation during DNA repair [6, 18-20]. Besides, multiple
independent studies demonstrated potential of SCR7 in improving precise genome editing
[
20-28]. A 2 to 19-fold increase in precise genome editing was reported both ex vivo and in
vivo, when SCR7 was used in tandem with CRISPR-Cas9 constructs. Since SCR7 blocked
NHEJ mediated repair of Cas9 cleaved chromosomal DNA, precise DSB repair through HR
was facilitated.
In the present study, we report that parental SCR7 can get autocyclized to a stable
form of SCR7 (SCR7-cyclized) possessing same molecular weight, molecular formula and
melting point. This form, upon oxidation can result in SCR7-pyrazine, with a different
molecular weight, molecular formula and structure. SCR7-cyclized and SCR7-pyrazine
exhibit inhibition of NHEJ in a Ligase IV dependent manner in vitro, and are efficient in
inducing cytotoxicity in cancer cell lines.
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RESULTS
Characterization of SCR7-cyclized and SCR7-pyrazine
In a previous study, we described a small molecule inhibitor, SCR7, that targets the
DNA binding domain of Ligase IV, thereby inhibiting the NHEJ pathway [17]. The described
structure of SCR7 (C H N OS) consists of two adjacent imine groups making it less stable
18
14
4
(
(
SCR7-parental) leading to spontaneous cyclization to give a more stable form of SCR7
SCR7-cyclized) (Fig. 1A). Several lines of experimentation suggest that reaction of 5,6-
diamino-4-hydroxy-2-mercaptopyrimidine with benzaldehyde gives rise to parental SCR7
5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol] as an intermediate.
[
Autocyclization of parental SCR7 could result in cyclized form of SCR7 (2-mercapto-6,7-
diphenyl-7,8-dihyropteridin-4-ol), which upon oxidation leads to the formation of SCR7-
pyrazine (2-mercapto-6,7-diphenylpteridin-4-ol) (Fig. 1A).
Incidentally, parental SCR7 and its stable autocyclized form (referred to as SCR7-
cyclized, henceforth) are structural isomers and they possess identical chemical formula
(
C H N OS), molecular weight, exact mass and same number of protons. We observed
18 14 4
o
that like parental SCR7, cyclized form of SCR7 possessed a melting point of 221-225 C,
o
while it was 194-196 C for SCR7-pyrazine. Pyrazine version of SCR7 was formed upon
dehydrogenation of SCR7-cyclized and possessed a distinct structure with an exact mass of
3
33.0798, molecular weight (332.07) and chemical formula (C H N OS).
18 12 4
We synthesized SCR7-cyclized and SCR7-pyrazine through a two-step facile
process (Fig. 1B). SCR7-cyclized was obtained with moderate yield in the presence of acetic
acid as an additive and DMF as a solvent. Good yield of the oxidative product SCR7-
pyrazine was obtained using nitrobenzene as an oxidizing agent and solvent (Fig. 1B).
SCR7-cyclized and SCR7-pyrazine were characterized by physical and spectroscopic
techniques. SCR6 (L189) was synthesized as described previously [17] and showed singlet
peaks at δ11.97, 11.74 and 9.63 ppm for OH, S-H, CH=N protons, respectively and NH2
1
peak was observed at δ 6.69 ppm in the H NMR spectrum.
1
13
Structural elucidation of SCR7-cyclized was carried out by H NMR and C NMR
spectroscopy (Fig. S1-3). The disappearance of peak at δ 9.63 and appearance of peak at δ
1
13
7
.49 (s, 1H, NH) & 6.00-6.01 (d, 1H, J = 2.4 Hz, benzylic CH) in H NMR and C signal at δ
2.6 indicates the 6π electrocyclic ring closure reaction to produce SCR7-cyclized. The
5
structure of SCR7-cyclized was further confirmed by D O exchange. We observed the
2
disappearance of peak at δ 7.49 and change in the splitting pattern of peak at δ 6.013-6.007
from doublet to singlet indicating the presence of NH adjacent to benzylic CH proton (data
1
13
not shown). Structural elucidation of SCR7-pyrazine was carried out by H NMR, C NMR
spectroscopy and HRMS (Fig. S4-6). Both NH and benzylic proton peaks were not observed
1
in the H NMR spectra of SCR7-pyrazine, which was obtained under mild oxidation
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conditions. Thus, SCR7-pyrazine showed disappearance of peak at 7.49 and 6.01-6.00
indicating the dehydrogenation of SCR7-cyclized.
SCR7-cyclized and SCR7-pyrazine inhibit DNA end joining
Previously, we have shown that testicular extracts from mice and rats are proficient in
classical NHEJ, and was dependent on Ligase IV, KU70 and KU80 [15, 29-32]. Hence, cell-
free repair assay system derived from rat testicular cells was used for comparing the effect
of inhibitors on NHEJ (Fig. 1C-G). Results showed that both SCR7-cyclized and SCR7-
32
pyrazine inhibited DNA end joining of [γ- P] ATP oligomeric labeled DNA irrespective of
type of DSB used (5’ compatible and two different 5’-5’ noncompatible DNA ends) (Fig. 1C-
G). While SCR7-cyclized showed significant inhibition from a concentration of 100 μM
onwards, SCR7-pyrazine was effective from 200 μM (Fig. 1C-G). End-joining inhibition
mediated by SCR7-cyclized and SCR7-pyrazine was also compared in a plasmid DNA
based assay system (Fig 1H,I). EcoRI digested pUC18 plasmid DNA was subjected to end-
joining reactions using rat testicular extracts in the presence of increasing concentrations of
the respective inhibitors (0.1, 0.5, 1 and 2 mM). Results showed a significant inhibition of
end-joining by both, SCR7-cyclized and SCR7-pyrazine at comparable concentrations (Fig.
1H,I). Taken together, different forms of SCR7 inhibited DNA-end joining reactions in vitro.
DNA Ligase IV is the preferred target for SCR7-cyclized and SCR7-pyrazine
Ligase IV/XRCC4 is the critical enzyme responsible for recreation of phosphodiester
bonds during NHEJ [33, 34]. To evaluate whether SCR7-cyclized and SCR7-pyrazine inhibit
Ligase IV mediated joining, purified Ligase IV/XRCC4 was used for joining assay [17].
Results showed that although both inhibitors blocked Ligase IV mediated break joining, the
effect was more pronounced when SCR7-cyclized was used as compared to SCR7-pyrazine
(
Fig. 2A). Importantly, ITC studies using Ligase IV/XRCC4, overexpressed and purified from
Baculovirus-infected insect cells, showed Kd values of 2.35 ± 0.76 µM and 0.5 ± 0.17 µM,
when incubated with SCR7-cyclized and SCR7-pyrazine, respectively (Fig. 2B). However, a
previously described Ligase inhibitor, L189 [35] did not result in any stable binding (Fig. 2B).
The specificity of inhibitors was further tested against purified Ligase I, Ligase III and T4
DNA Ligase (Fig. 2C-E). Results showed that neither SCR7-cyclized nor SCR7-pyrazine
showed any remarkable inhibition on joining catalyzed by Ligase I, Ligase III or T4 DNA
ligase (Fig. 2C-E).
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SCR7-cyclized and SCR7-pyrazine inhibit V(D)J recombination inside cells
NHEJ is responsible for repair of DNA double-strand breaks generated during V(D)J
recombination, the process by which antibody diversity is generated [36, 37]. Therefore, we
examined the impact of Ligase IV inhibitors on V(D)J recombination using previously
described extrachromosomal assay system (Fig. 3A) [38-41]. Since both the chemicals
induced cell death upon treatment in Nalm6 cells, comparable number of ampicillin colonies
were analysed in both treated and vehicle control samples. While SCR7-cyclized treatment
resulted in 2.1-fold reduction in coding joint formation, reduction was only 1.5-fold in the case
of SCR7-pyrazine upon transfection with pGG51 in pre-B cells (Nalm6) (Fig. 3B). Significant
reduction in signal joint formation was also observed (data not shown). Thus, both SCR7-
cyclized and SCR7-pyrazine can block NHEJ during V(D)J recombination within the cells,
although SCR7-cyclized was more efficient.
Treatment with SCR7-cyclized and SCR7-pyrazine leads to unrepaired DNA breaks
inside the cells
Based on above results, we tested whether treatment with Ligase IV inhibitors could
block chromosomal NHEJ resulting in accumulation of unrepaired breaks within the cells.
HeLa cells were treated with increasing concentrations (50, 100 μM for 24 h) of SCR7-
cyclized and SCR7-pyrazine, and presence of DSBs was determined using γ-H2AX or
53BP1 foci analysis. Results showed significant increase in γ-H2AX and 53BP1 foci
formation upon treatment with SCR7-cyclized and SCR7-pyrazine in a concentration
dependent manner (Fig. 4A-D), suggesting inhibition of endogenous NHEJ within the cells.
Further, comet assay following treatment with SCR7-cyclized and SCR7-pyrazine in Nalm6
cells (100, 250 μM for 24 h) showed a concentration dependent increase in the tail moment
of comets (Fig. 4 E,F), confirming the accumulation of DNA breaks following treatment with
SCR7-cyclized and SCR7-pyrazine.
SCR7-cyclized and SCR7-pyrazine can potentiate the effect of γ-radiation leading to
enhanced number of DSBs
Our previous studies indicate that SCR7 can potentiate both chemo and radiotherapy
[
17]. Based on this, we examined whether SCR7-cyclized and SCR7-pyrazine treatment
can cause increased levels of radiation-induced DNA breaks, when coadministered, as they
inhibit NHEJ in irradiated cells. To test this, HeLa cells were irradiated with γ-rays (1 Gy), or
treated with SCR7-cyclized and SCR7-pyrazine form (100 μM) or coadministered with both.
Cells were harvested after 1 h post treatment and evaluated for γ-H2AX and 53BP1 foci
formation. Results showed significantly increased levels of γ-H2AX foci, upon
coadministration with SCR7-cyclized or SCR7-pyrazine, compared to IR treatment alone
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(
Fig. 5A,B). Similarly, we also observed significantly high levels of 53BP1 foci, when IR was
coadministered with SCR7-cyclized or SCR7-pyrazine (Fig. 5C,D). These results suggest
that both SCR7-cyclized and SCR7-pyrazine can block residual NHEJ in IR treated cells
leading to increased levels of unrepaired DSBs. Further, we performed a time-course
analysis for investigating the repair kinetics of IR-induced DSBs upon coadministration of
radiation and inhibitors. Cells were assessed for 53BP1 foci formation at 1, 2, 6 and 12 h
recovery period post IR (Fig. 6A-C). Results showed a time-dependent decrease of 53BP1
foci formation in case of radiation alone (post 2 h), suggesting active DSB repair in these
cells post irradiation (Fig. 6A-D). Interestingly, treatment with SCR7-cyclized or SCR7-
pyrazine alone showed a steady increase in the number of 53BP1 foci within cells (Fig. 6D)
suggesting inhibition of endogenous NHEJ in a time-dependent manner leading to
accumulation of DSBs. However, coadministration of IR with SCR7-cyclized or SCR7-
pyrazine resulted in significant reduction in repair kinetics over time, as compared to
samples treated with IR alone (Fig. 6D). Fold change analysis showed that while there was
3.4-fold decrease in number of 53BP1 foci in samples treated with radiation alone at 12 h
when compared to 2 h, coadministration of SCR7-cyclized or SCR7-pyrazine with radiation
showed only 1.4- and 1.6-fold decrease, respectively (Fig. 6E). Taken together, our results
reveal that coadministration of these inhibitors with radiation leads to abrogation of NHEJ-
mediated repair process, thus ultimately leading to accumulation of unrepaired DSBs in
treated cells.
Different forms of SCR7 induce cytotoxicity in a Ligase IV dependent manner
Previously, we have observed varying levels of cytotoxicity induced by SCR7 in
different cancer cell lines [17]. Therefore, we tested the ability of SCR7-pyrazine (0, 50, 100
and 250 μM for 48 h) in inducing cell death in three different cell lines, MCF7, HeLa and
Nalm6. Results showed that SCR7-cyclized and SCR7-pyrazine induced cytotoxicity in a
concentration dependent manner, suggesting elevated DSBs following the compound
treatment, ultimately leading to cell death (Fig. 7A-C).
In order to evaluate Ligase IV and hence, NHEJ-dependence during cytotoxicity
induced by SCR7-cyclized and SCR7-pyrazine, we evaluated their sensitivity in Ligase IV
genetic knockout cells, which are deficient in NHEJ [34]. Knock out cell line, N114 and its
wild type, Nalm6 cells were treated with increasing concentrations of SCR7-cyclized and
SCR7-pyrazine (0, 50, 100 and 250 μM for 48 h) and evaluated for cytotoxicity (Fig. 7D,E).
Results showed that unlike wild type cells, N114 cells did not exhibit any cytotoxicity at 50
and 100 μM concentrations of SCR7-cyclized. Even at the highest concentration used (250
μM), there was no significant cell death (Fig. 7D). However, in the case of SCR7-pyrazine
treated samples, we observed significant cell death at 250 μM treated samples, indicating
Ligase IV independent cell death at higher concentrations (Fig. 7E). This indicates that the
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observed elevated cytotoxicity in the case of SCR7-pyrazine could be contributed by Ligase
IV independent mechanism. Thus, although cytotoxicity induced by SCR7-cyclized was due
to the presence of Ligase IV, cell death elicited by SCR7-pyrazine may not be entirely
dependent on Ligase IV.
Next, we examined whether the increased levels of DSBs in the IR and NHEJ
inhibitor coadministered cells could result in elevated cell death. To evaluate this, Nalm6 and
HeLa cells were co-treated with IR (1 Gy) and SCR7-cyclized or SCR7-pyrazine (100 μM)
and cell death was monitored using trypan blue assay (Fig. 8A,B). Results showed that
similar to SCR7-cyclized, SCR7-pyrazine potentiated the effect of radiation significantly, in
both the cell lines tested (Fig. 8A,B). Therefore, our data suggest that increased DSBs
following coadministration of IR and NHEJ inhibitors resulted in elevated cytotoxicity in the
tested cancer cell lines.
DISCUSSION
Parental SCR7 and SCR7-cyclized possess same molecular weight and formula,
unlike SCR7-pyrazine
In a recent study, we had described a small molecule inhibitor, SCR7 that blocks
NHEJ, resulting in accumulation of broken DNA within cells, thereby causing tumor
regression in mice [17]. In-depth evaluation suggested that parental SCR7, which is less
stable, can get spontaneously cyclized into SCR7-cyclized and subsequently oxidized to
SCR7-pyrazine.
Both parental SCR7 and SCR7-cyclized have same molecular mass, molecular
weight and melting point. Besides, they also possess the same number of protons. However,
unlike parental SCR7 and SCR7-cyclized, SCR7-pyrazine has a distinct mass, melting point,
number of protons, molecular formula and chemical structure. It appears that reaction
conditions, such as temperature, pH and purification strategies can facilitate generation of
different forms. A similar scenario, wherein three different forms of Mirin, an inhibitor of MRN
complex, with same mass were reported previously [42, 43].
In a recent report, another cyclized form of SCR7, named as SCR7-R was described
o
[
44, 45]. However, this form of SCR7 had a melting point of 273-280 C, and molecular
weight of 333.0959, which was different compared to SCR7-parental, SCR7-cyclized and
SCR7-pyrazine.
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SCR7-cyclized and SCR7-pyrazine inhibit NHEJ in a Ligase IV dependent manner with
varying efficiencies
Our studies revealed that similar to SCR7-cyclized, treatment with SCR7-pyrazine
also resulted in inhibition of DNA end joining in a concentration dependent manner.
Importantly, SCR7-pyrazine also inhibited joining of different 5’-5’ noncompatible ends,
which requires processing of DNA breaks prior to ligation, and is dependent on classical
NHEJ proteins [29, 31]. This was also consistent with the observed inhibition of NHEJ by
SCR7 [17, 18, 46-49]. Besides, similar to SCR7-cyclized, SCR7-pyrazine also showed
specific inhibition of joining catalyzed by Ligase IV and their effect on Ligase III, Ligase I and
T4 DNA ligase mediated joining was minimal in vitro.
During V(D)J recombination, DNA breaks generated at recombination signal
sequence by RAGs are repaired through NHEJ [36, 37]. Upon evaluation of coding joint
formation using episomal DNA substrates, we observed that addition of SCR7-cyclized or
SCR7-pyrazine significantly reduced recombination frequency. A comparable effect was also
observed during signal joint formation; however, among the inhibitors, SCR7-pyrazine was
less effective. Hence, SCR7-cyclized and SCR7-pyrazine are potent and effectively inhibited
NHEJ in a Ligase IV dependent manner.
Inhibition of NHEJ within the cells can result in accumulation of DNA double-strand
breaks [17]. We observed that treatment with SCR7-pyrazine resulted in elevated levels of γ-
H2AX foci, which was comparable to that of SCR7-cyclized suggesting unrepaired DSBs.
The increased levels of comets seen following SCR7-cyclized and SCR7-pyrazine treatment
confirmed the accumulation of DNA breaks within the cells. Previously, we showed that
accumulation of DNA breaks could result in cell death [17]. Consistent to that we observed
that treatment with both SCR7-cyclized and SCR7-pyrazine culminated in cell death in all the
cell lines tested. Interestingly, the sensitivity of cell lines varied and exhibited different IC50
values. Therefore, it is important to point out that various cell lines may exhibit differential
sensitivity towards different forms of SCR7. Varying levels of Ligase IV expression, nature of
cell type etc. could be the factors responsible for it.
The Ligase IV genetic knockout of Nalm6 cells showed no cell death following SCR7-
cyclized treatment, while robust cytotoxicity was seen in wild type cells. In contrast, there
was significant cell death at high concentrations upon SCR7-pyrazine treatment even in
Ligase IV knockout cells, although it was much less compared to wild type cells. This reveals
that although cytotoxicity induced by SCR7-cyclized is Ligase IV-dependent, SCR7-pyrazine
induced toxicity could be attributed to other targets as well, particularly at high
concentrations.
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SCR7-cyclized and SCR7-pyrazine could potentiate radiotherapy
Since NHEJ inhibitors can block residual DSB repair following irradiation, this is
considered as an ideal strategy to improve the efficacy of radiotherapy and in some
instances, chemotherapy. Previously, we showed that SCR7 could improve radio and
chemosensitivity in mice tumor models and cancer cell lines [17, 50, 51]. Interestingly, we
noted that treatment with SCR7-cyclized or SCR7-pyrazine along with radiation resulted in
elevated levels of DSBs, which was higher than either of the agents alone. Further,
administration of both the chemicals in combination with radiation resulted in a synergistic
effect leading to enhanced cell death. Thus, it appears that similar to cyclized form of SCR7,
even its oxidized form can potentiate radiotherapy. In a quantitative study, we observed that
cyclized form of SCR7 can bring down the effective dose of radiation by 50-75% in mice
(
SV/SCR, unpublished). Recently, it has also been reported that SCR7 could improve the
efficacy of melphalan by 4 to 7-fold in myeloma patient primary cells [46]. Our studies reveal
that SCR7 improves efficacy of temozolomide treatment in glioma primary patient cells
several fold (VK/SCR, unpublished). Impact of SCR7-pyrazine in primary patient cells of
various cancer types needs to be investigated further.
SCR7 as a tool to improve precise genome editing
In recent years, CRISPR-Cas has become an effective tool for targeted genome
editing [20, 52-54]. The DSBs generated by the endonuclease activity of Cas9 results in
activation of DSB repair machinery, that can mend the breaks by either NHEJ or HR. NHEJ
being an error-prone process can introduce indels (insertions and deletions) at the joining
site, thereby disrupting the target locus. Since homology directed repair (HDR) is efficient in
NHEJ-deficient cell lines [55], inhibiting NHEJ can lead to upregulation of HDR, and hence
effective precise genome editing.
Recent studies have discovered a novel application of SCR7 in CRISPR-Cas
mediated genome editing [20, 21, 23]. SCR7 treatment resulted in inhibition of NHEJ,
thereby increasing the efficiency (~2-19 fold) of HR-mediated precise genome editing [21,
23, 26]. Considering that both SCR7-cyclized and SCR7-pyrazine could result in inhibition of
NHEJ, although with varying efficiencies, they can be harnessed as excellent tools for
CRISPR-Cas mediated genome editing. Recent studies have reported the successful use of
SCR7 in CRISPR-Cas mediated gene editing in Zebra fish, wherein an increased HDR rate
of ~74% was achieved with the combined use of SCR7 and RS-1, a previously reported
RAD51 stimulatory compound [56, 57] . It may be interesting to extend their application to
other model organisms such as Drosophila and C. elegans where CRISPR-Cas has been
employed.
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In conclusion, parental SCR7 can get autocyclized into a stable form, SCR7-cyclized,
having the same molecular weight, molecular mass, melting point and number of protons.
SCR7-cyclized upon further oxidization results into SCR7-pyrazine having different
molecular weight and melting point. Importantly both the forms can inhibit NHEJ in a Ligase
IV-dependent manner. Further, the treatment results in accumulation of DSBs inside cells,
finally leading to cell death. However, SCR7-cyclized is more specific in its action inside the
cells. Since the molecular weight of SCR7-pyrazine (332.07) is less than that of SCR7-
cyclized and SCR7-parental (334.09), and SCR7-cyclized is the stable form compared to
SCR7-parental, it is evident that the SCR7 used in previous studies described in the
literature by different groups demonstrating its effect in genome editing, cancer therapeutics
and as a biochemical inhibitor of DSB repair, is most likely the cyclized version.
Materials and methods
Enzymes, chemicals, and reagents
Chemicals and reagents used in the study were purchased from Sigma Chemical Co.
(
St. Louis, MO, USA) and Sisco Research Laboratories (Andheri (E), Mumbai, India).
Restriction enzymes and other DNA-modifying enzymes were obtained from New England
Biolabs (Beverly, MA, USA) and Fermentas (Glen Burnie, MD, USA). Culture media were
from Lonza (Walkersville, MD, USA), and Himedia (Bengaluru, Karnataka, India). Foetal
bovine serum and PenStrep were from Gibco BRL (Gaithersburg, MD, USA) and MP
Biomedicals (Santa Ana, CA, USA). Radioisotope-labelled nucleotides were from BRIT (Navi
Mumbai, Maharashtra, India). Antibodies were purchased from Santa Cruz Biotechnology
(
Dallas, TX, USA), Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA,
USA).
Plasmids
Plasmid encoding cDNA of human Ligase IV (NCBI accession number BC037491)
was purchased from Open Biosystems (Lafayette, CO, USA). Plasmid coexpressing Ligase
IV and XRCC4 was a kind gift from Mauro Modesti (France). pGG49 and pGG51 were from
M. Lieber, USA. Ligase I and Ligase IIIα/XRCC1 plasmids were from A. Tomkinson (USA)
and K. W. Caldecott (UK), respectively.
Cell lines and culture
HeLa (human cervical cancer), MCF7 (human breast cancer), Nalm6 (Pre B
leukemia) and N114 (Ligase IV knockout of Nalm6), were used for the study. HeLa and
MCF7 cells were grown in DMEM high glucose with L-glutamine containing 10% FBS while
Nalm6 and N114 cells were grown in RPMI medium containing 15% FBS. The cells growing
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in log phase were used for transfection [40, 58]. The media was supplemented with 100
μg/ml Penicillin G and streptomycin and incubated at 37ºC in a humidified atmosphere
containing 5% CO2.
Nalm6 and N114 [34] cells were from Dr. M. Lieber, USA. MCF7 was from National
Centre for Cell Science, Pune, India, and HeLa was from Dr. A. Karande, India.
Oligomeric DNA
Oligomers used in the study were synthesized from Eurofins Genomics (Bengaluru,
Karnataka, India) and IDT (San Jose, CA, USA). The oligomers were purified using 10-15%
denaturing PAGE, as described [59] (Table S1).
Preparation of oligonucleotide dsDNA substrates
32
The oligomeric DNA were 5' labelled with [γ- P] ATP using T4 Polynucleotide Kinase
(
see below). SCR19 was annealed to SCR20 or VK11 to generate 5'-5' compatible or 5'-5'
noncompatible substrates, respectively [30, 31]. SB1 was annealed to SB5, MP9 to MP10 to
generate noncompatible end substrates. In order to prepare the nicked DNA substrate,
radiolabelled MS68 was annealed to cold (5’ phosphorylated) MS69 and MS70 and
annealing was confirmed on a gel.
Chemistry
General information
All work related to analytical thin layer chromatography were performed with E.
Merck silica gel 60 F254 aluminium plates and were visualized under UV light. The following
mobile phases were employed for TLC: chloroform, methanol and hexane, and ethyl
acetate in different ratios. The instrumental techniques employed for the characterization of
1
13
1
the newly synthesized compounds include H and C NMR and mass spectroscopy. H
13
and C−NMR spectra were recorded on an Agilent WM (400 and 100 MHz) Fourier
transforms NMR spectrometer in CDCl or DMSO-d solvent using tetramethylsilane (TMS)
3
6
as internal standard. Chemical shifts were recorded in ppm relative to TMS. Mass and
purity were recorded on a LC–MSD-Trap-XCT (Agilent technologies Inc). All the reagents
and chemicals used were from Sigma Chemical Co. (St. Louis, MO, USA).
Synthesis of SCR6
SCR6 was synthesised as described before [17]. Briefly, a suspension of 5,6-
diamino-4-hydroxy-2-mercaptopyrimidine and benzaldehyde in dimethyl formamide and
acetic acid were stirred at room temperature for 16 h. Once the reaction was completed, the
reaction mixture was added slowly to ice cold water, solid was precipitated out and collected
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by vacuum filtration, washed with water and compound was recrystallized from dimethyl
1
formamide-ethanol to get pure compound, SCR6. Yield−0.93 g (60%); H NMR (400 MHz,
DMSO-d ): δ 11.97 (s, 1H), 11.74 (s, 1H), 9.63 (s, 1H), 7.86 (d, J=7.2 Hz, 2H), 7.40−7.32 (m,
6
3
H). 6.69 (s, 2H, NH ).
2
Synthesis of SCR7-cyclized
A suspension of SCR6 (0.93 g, 0.0037 mol) and benzaldehyde (0.4 g, 0.0037 mol) in
dimethyl formamide (30 mL) and acetic acid (3 mL) was heated at 200ºC for 8-10 h. Once
the reaction was completed, the reaction mixture was added slowly to ice cold water and a
solid was precipitated out which was collected by vacuum filtration, washed with water and
compound was recrystallized from dimethyl formamide-ethanol to get pure compound.
o
1
Yield−0.44 g (35%); Melting point: 221-225 C; H NMR (400 MHz, DMSO-d ): δ 12.03 (s,
6
1
2
1
3
H), 11.96 (s, 1H), 7.82−7.80 (m, 2H), 7.49 (s, 1H, NH), 7.38−7.24 (m, 8H), 6.01-6.00 (d, J =
13
.4 Hz, 1H, CH); C NMR (100 MHz, DMSO-d ): 173.4, 157.8, 146.9, 143.4, 140.5, 136.5,
6
+
30.0, 129.5, 128.9, 128.8, 127.2, 126.7, 104.6, 52.6. m/z (M+H) calcd for C H N OS
18
14
4
35.08. found: 335.10.
Synthesis of SCR7-pyrazine
Oxidation of SCR7-cyclized (0.44 g, 0.0013 mol) was carried out in nitrobenzene by
refluxing for 24 h. After completion of reaction, solids were separated which was collected by
vacuum filtration, washed several times with diethyl ether to get pure SCR7-pyrazine.
o
1
Yield−0.34 g (80%); Melting point: 194-196 C; H NMR (400 MHz, DMSO-d ): δ 13.40 (s,
6
1
3
1
1
+
H), 12.80 (s, 1H), 7.40−7.31 (m, 10H); C NMR (100 MHz, DMSO-d ): δ 176.1, 158.8,
6
56.2, 149.4, 147.3, 138.0, 137.5, 130.1, 130.11, 129.9, 129.1, 128.6, 128.6, 127.5. m/z (M
+
H) Calcd. for C H N OS 333.07. found: 333.09.
18
12
4
5′ end-labelling of oligomers
5′ end-labelling of oligomeric DNA was performed using T4 polynucleotide kinase
32
o
and [- P] ATP at 37 C for 1 h as described [10, 58]. Labelled substrates were purified using
o
Sephadex G-25 column and stored at -20 C till use. Radiolabelled duplex DNA was
prepared by annealing labelled strand with 5 fold excess of complementary unlabelled strand
in 100 mM NaCl and 1 mM EDTA in a boiling water bath, followed by gradual cooling, as
described earlier [10, 58].
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Ethics statement
Rats were maintained as per the principles and guidelines of the ethical committee for
animal care of Indian Institute of Science in accordance with Indian National Law on animal
care and use. The experimental design of the present study was approved by Institutional
Animal Ethics Committee (Ref. CAF/Ethics/228/2011), Indian Institute of Science,
Bangalore, India.
Preparation of cell-free extracts
Rat testicular extract was prepared as described earlier [31, 32]. In brief, testes were
collected from male Wistar rats, Rattus norvegicus (4–6 weeks old), minced to prepare
single cell suspension, and counted using a haemocytometer. Approximately, 2 x
7
1
0 cells/ml were pelleted, resuspended in hypotonic buffer (buffer A: 10 mM Tris–HCl (pH
8.0), 1 mM EDTA, 5 mM DTT and 0.5 mM PMSF), and homogenized with protease
inhibitors (1 μg/ml each of leupeptin, aprotinin, and pepstatin). An equal volume of buffer B
50 mM Tris–HCl (pH 8.0), 10 mM MgCl , 2 mM DTT, 0.5 mM PMSF, 25% sucrose and
(
2
50% glycerol) was added followed by addition of neutralized, saturated ammonium sulfate
solution (11% cut off). Supernatant was then collected after centrifugation (3 h at 32,000 g
at 2°C), proteins were precipitated by ammonium sulfate (65%) method and dialyzed (buffer
C: 25 mM HEPES–KOH (pH 7.9), 0.1 M KCl, 12 mM MgCl , 1 mM EDTA, 2 mM DTT and
2
17% glycerol) for 16 h. Extracts were aliquoted, snap-frozen in liquid nitrogen and stored at
−
80°C until use.
Expression and purification of proteins
Ligase IV/XRCC4, Ligase IIIα/XRCC1 and Ligase I were overexpressed in bacteria
and purified as described before [17, 60]. Purity and identity of the proteins were confirmed
using SDS-PAGE and western blot analysis.
In vivo Recombination assay
The human lymphoid cell line, Nalm6 was cultured and transfected with episomal
constructs pGG51 (coding joint) or pGG49 (signal joint) along with SCR7-cyclized or SCR7-
pyrazine (100 µM) by electroporation (300V, 900 µF, ∞ Ω, 4 mm cuvette thickness) and
o
incubated for 48 h at 37 C [38-41]. Equivalent amount of DMSO served as vehicle control.
The DNA was recovered using Hirt harvest method and the reaction products were
transformed in E. coli DH10B and selected on ampicillin (A) and chloramphenicol-ampicillin
(
CA) LB agar plates to estimate recombination frequency, as described previously [38-41].
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DNA end-joining reaction
NHEJ assay was performed as described before [30, 31, 61-64]. Briefly, cell-free
extracts prepared from rat testes were incubated with or without SCR7-cyclized or SCR7-
o
pyrazine (100, 200, 500 µM and 1 mM) for 30 min at 25 C in NHEJ buffer (25 mM Tris-HCl,
[
pH 7.5], 75 mM NaCl, 10 mM MgCl , 42.5 mM KCl, 0.025% Triton X-100, 100 µg/ml BSA,
2
10% PEG, and 5% glycerol). Similarly, purified Ligases were incubated with or without
o
SCR7-cyclized/ SCR7-pyrazine (50, 100, 200, 500 µM and 1 mM) for 30 min at 25 C.
32
Further, γ-[ P] ATP labelled DNA oligomeric substrate (4 nM) was added to the reaction mix
o
and incubated for 1 h at 25 C. Reaction was terminated by adding 10 mM EDTA, followed by
extraction of DNA products using phenol-chloroform and precipitation method. Purified
products were then resolved on a 8% denaturing PAGE, followed by drying and exposing the
gel to a PhosphorImager screen. Signals were detected using PhoshorImager (FLA9000,
Fuji, Japan) and the data analyzed on Multigauge software (V3.0). The intensity of joined
products was quantified and represented as PSLU (Photo-stimulated luminescence units).
Protein purification of Ligase IV/XRCC4 in insect cells
Full length Ligase IV tagged with ten histidines and full length XRCC4 were co-
2+
expressed in Sf21 insect cells [65]. The complex was purified by immobilized Ni ion affinity
chromatography (GE Healthcare), followed by anion exchange chromatography (Resource
Q). Purified proteins were dialyzed in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM beta-
mercaptoethanol, 5% glycerol and stored at -80ºC. Protein concentrations are expressed in
molarity of 1:2 complexes for Ligase IV/XRCC4.
Ligase IV/XRCC4 interactions with inhibitors by microcalorimetry
Experiments were performed on a VP-ITC calorimeter (Microcal), as described
before [66, 67]. All solutions were degassed under vacuum. The reaction cell (1.8 ml) was
loaded with between 6 and 16 μM Ligase IV/XRCC4 solutions. The syringe (500 µl) was
filled with SCR7-cyclized, SCR7-pyrazine, L189 at concentrations between 60 and 160 µM.
For these experiments, L189 was purchased from Tocris Bioscience (3561). Control
experiments were performed with SCR7-cyclized solutions injected into buffer.
Thermodynamic parameters ΔH, N, and Ka were obtained by nonlinear least-squares fitting
of the experimental data using the single set of independent binding sites model of the Origin
software provided with the instrument. The free energy of binding (ΔG) and the entropy (ΔS)
were determined using the classical thermodynamic formulas: ΔG = −RT ln(Ka) and ΔG =
ΔH − TΔS. All binding experiments were performed in duplicate at 25°C.
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Immunofluorescence
o
Cells (25,000/ml) were grown on coverslips for 24 h at 37 C, followed by treatment
with either SCR7-cyclized or SCR7-pyrazine (50 and 100 µM), and processing for
immunofluorescence as described before [17, 51, 64]. Briefly, after 24 h treatment, cells
were harvested, fixed (4% paraformaldehyde for 10 min), permeabilized (PBS containing
0.1% Triton X-100 for 5 min) and blocked (PBST containing 0.1% BSA and 10% foetal
bovine serum (FBS) for 1 h). Cells were then incubated with anti-γH2AX antibody (1:500;
Millipore) or 53BP1 antibody (1: 200; SantaCruz Biotechnologies, USA) overnight at 4°C,
washed with PBST and incubated with corresponding biotinylated secondary antibody
(
1:200) for 2 h. Cells were then washed, incubated with streptavidin-FITC (1:500) for 20 min.
The nucleus was counterstained using DAPI/Propidium Iodide, mounted using Glycerol and
antifade DABCO, and images were captured using Zeiss Apotome Fluorescence Microscope
(
Germany) and Zeiss Laser Confocal microscope (Germany).
Experiments were also performed following cotreatment with IR (1 Gy) and SCR7-
cyclized or SCR7-pyrazine (100 µM for 1 h) and evaluated post staining with 53BP1 and γ-
H2AX antibodies for foci formation. For time-course studies post irradiation, cells were
harvested at 1, 2, 6 and 12 h post IR, SCR7-cyclized, SCR7-pyrazine or combination
treatment. Cells were fixed using 2% paraformaldehyde and processed for IF using anti-
53BP1, as described above.
Neutral comet assay
Comet assay was performed as described previously [17, 51]. Briefly, Nalm6 cells
25,000/ml) were seeded, treated with SCR7-cyclized or SCR7-pyrazine (100 and 250 µM)
(
for 24 h, harvested post treatment and washed with PBS, followed by addition of low melting
agarose. The mixture was then spread onto a glass slide and submerged in neutral lysis
o
buffer overnight at 37 C. Slides were then subjected to electrophoresis at 12 V for 25 min,
followed by staining with Propidium Iodide. A minimum of 50 cells were imaged per sample
for each experiment, scored for comets using Comet Score software and analyzed for %
DNA in tail, tail moment and olive moment. Images were captured using Zeiss Apotome
Fluorescence Microscope (Germany).
Cytotoxicity assay
Cytotoxicity was assessed using the Trypan Blue dye exclusion assay as described
before [68-72]. Briefly, HeLa, MCF7, Nalm6 and N114 cells (25,000/ml) were seeded and
treated with SCR7-cyclized or SCR7-pyrazine (50, 100 and 250 µM for 48 h). Cells were
harvested, stained with Trypan blue dye, followed by counting of viable cells. Each
experiment was repeated a minimum of three times. Experiments were also performed
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following cotreatment with IR (1 Gy) and SCR7-cyclized or SCR7-pyrazine (100 µM for 48 h)
and evaluated for cytotoxicity.
Statistical analysis
The results are expressed with standard error mean. All analyses were done with the
GraphPad software using one-way ANOVA or Student's t-test. Statistical significance was
considered as ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001.
Acknowledgements
We thank Sheetal Sharma and members of SCR laboratory for discussion and
comments on the manuscript. We thank central animal facility and confocal facility, IISc for
their help. This work was supported by grants from Centre for the Promotion of Advanced
Research [Grant No. IFC/5203-4/2015/131] to SCR and JBC, and financial assistance from
IISc-DBT partnership programme [DBT/BF/PR/INS/2011-12/IISc] to SCR. JBC is supported
by ARC program (SLS220120605310), by ANR (ANR-12-SVSE8-012) and by INCA
DomRep (PLBIO 2012-280). SV is supported by Senior Research fellowship from IISc, India,
and VG by Senior Research fellowship from CSIR, India.
Conflict of interest
Authors disclose that there is no conflict of interest.
Author contributions
SCR, MK and BC conceived the project and provided reagents. SCR, MK, JBC, SVV,
SW and VKG designed the experiments. SVV, VG, VR, MN, RK, NK, UR, GR, DD, MP, BC,
HA and MS conducted the biology experiments. SWA, VKG, FJ, KSR conducted the
chemistry experiments. SCR, MK, BC, JBC, SSK, SVV, SWA and VKG interpreted the data.
SCR, MK and SVV wrote the manuscript.
This article is protected by copyright. All rights reserved.

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Figure 1. Chemical structures of different forms of SCR7 and their effect on end
joining of DNA double-strand breaks. A. Parental SCR7 upon autocyclization can get
converted into a stable form of SCR7 (SCR7-cyclized), which following dehydrogenation is
converted into SCR7-pyrazine. B. Synthesis of SCR7-cyclized and SCR7-pyrazine. C.
Representative PAGE profile showing effect of SCR7-cyclized and SCR7-pyrazine on joining
of DSBs with 5'-5' compatible termini catalyzed by rat testicular extracts (n=4). 'M' represents
50 bp ladder. D. Bar graph depicting DNA end-joining efficiency upon treatment with SCR7-
cyclized and SCR7-pyrazine for 5'-5' compatible DNA substrate. E. PAGE profile showing
effect of SCR7-cyclized and SCR7-pyrazine on joining of a 55 bp DNA substrate possessing
5'-5' noncompatible ends catalyzed by rat testicular extracts. F. Bar graph showing
quantification of joining efficiency for 5'-5' noncompatible end substrates as determined
using MultiGauge based on multiple gels (n=3). G. Bar graph showing quantification of
joining efficiency of a 75 bp DNA substrate possessing 5'-5' noncompatible ends. H.
Representative gel image showing inhibition of DNA end-joining by SCR7-cyclized and
SCR7-pyrazine when a linear (EcoRI) plasmid DNA was used as a substrate. pUC18
plasmid DNA was digested using EcoRI to generate compatible ends. Rat testicular extract
was incubated with increasing concentrations of SCR7-cyclized and SCR7-pyrazine (0.1,
o
0
.5, 1 and 2 mM) for 30 min at 25 C. Linear pUC18 DNA substrate was then added and the
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o
reaction was allowed to proceed for 1 h at 25 C. Joined products were purified and resolved
on a 1% agarose gel. I. Bar graph depicting quantification of joined products as quantified
using MultiGauge software. Each experiment was repeated (n=3), joined product intensity
quantified, and presented as bar graphs. In all panels, bar diagram represents mean ± SEM
(
ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In all cases, significance was
calculated using the Student’s t-test in GraphPad Prism software.
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Figure 2. Analysis of specificity of SCR7-cyclized and SCR7-pyrazine against different
mammalian DNA Ligases. A. Effect of SCR7-cyclized and SCR7-pyrazine on DNA joining
catalyzed by purified DNA Ligase IV/XRCC4 on a DNA substrate bearing nick. In each case,
50, 100, 200, 500 and 1000 µM inhibitor concentrations were tested. Each experiment was
repeated (n=3) and the joining efficiency was quantified and plotted as a bar graph (right
panel). B. Measurements of interactions by microcalorimetry (ITC). The interactions between
Ligase IV/XRCC4 and SCR7-cyclized, SCR7-pyrazine or L189 are shown. Thermograms
(
top) and isotherm of titrations (below) have been depicted for each titration. PAGE profile of
purified Ligase IV/XRCC4 (L4/X4) proteins purified from Baculovirus infected insect cells is
also shown. C. Denaturing PAGE profile showing effect of SCR7-cyclized and SCR7-
pyrazine on joining of nicked DNA catalyzed by Ligase I. Bar graph with error bars is shown
on the right. D. Effect of SCR7-cyclized and SCR7-pyrazine on DNA joining catalyzed by
Ligase III/XRCC1. Bar graph with error bars is also presented. E. Bar graph depicting effect
of SCR7-cyclized and SCR7-pyrazine on joining of double-stranded DNA catalyzed by T4
DNA Ligase. In each case (n=3), the joining efficiency is plotted as a bar graph showing
mean ± SEM (ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In all cases,
significance was calculated using the Student’s t-test in GraphPad Prism software.
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Figure 3. Evaluation of efficiency of V(D)J recombination upon treatment with SCR7-
cyclized or SCR7-pyrazine. A. Schematic presentation showing human V(D)J recombinase
assay. The diagram depicts transfection of episome into a human pre-B cell line, Nalm6,
active for V(D)J recombination, in presence of SCR7-cyclized or SCR7-pyrazine (100 μM).
DMSO treated cells served as vehicle control. After 48 h inside the cells, the episomes were
harvested by Hirt harvest method and transformed into E. coli DH10B for detection of
recombinants on LB agar plates containing ampicillin and chloramphenicol (Amp + Cam).
The recombination is depicted between a consensus 12- (grey triangle) and 23- (black
triangle) signal of pGG51, leading to coding joint formation. “cat” denotes the
chloramphenicol acetyl transferase gene, and “stop” denotes the prokaryotic transcription
terminator. The E. coli lac promoter is denoted as “P ”. B. Table summarizing effect of
lac
SCR7-cyclized and SCR7-pyrazine on V(D)J recombination in pre-B cells. The episome,
pGG51 was transfected into Nalm6 cell line, and the recombination efficiency was tested
following transformation into E. coli. The recombination frequency is calculated by the
formula: (CA/A)x100, where the number of colonies obtained on ampicillin plate (A) and
chloramphenicol-ampicillin (CA) selective media for the episomal substrate pGG51. Fold
change in recombination following treatment with SCR7-cyclized and SCR7-pyrazine is also
shown. Transfections were performed (n=3) followed by multiple transformations using each
batch.
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Figure 4. Detection of DNA double-strand breaks within cells upon treatment with
SCR7-cyclized and SCR7-pyrazine. A. Representative images showing γ-H2AX foci
formation in HeLa cells following treatment with SCR7-cyclized and SCR7-pyrazine (50 µM
and 100 µM) for 24 h. DMSO was used as a vehicle control. γ-H2AX is depicted by Alexa-
flour 568 staining (red), whereas nucleus is counterstained using DAPI (blue). Each
experiment was repeated independent times (n=3). The scale bar represents 0.2 µm. B. Bar
graph depicting γ-H2AX foci per cell representing mean ± SEM (ns: not significant, *p < 0.05,
**p < 0.005, ***p < 0.0001). In all cases, significance was calculated using the Student’s t-
test in GraphPad Prism software. A minimum of 100 cells were analyzed per batch for foci
formation, followed by calculation of average foci in each group. C. Representative images
depicting 53BP1 foci formation in HeLa cells upon treatment with SCR7-cyclized and SCR7-
pyrazine (50 µM and 100 µM). 53BP1 is stained with FITC (green), whereas nucleus is
counterstained using propidium iodide (red). The scale bar represents 0.2 µm. D. Bar graph
showing average 53BP1 foci in each group upon treatment with SCR7-cyclized and SCR7-
pyrazine. E. Representative images showing direct double-strand break formation in Nalm6
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cells as assessed using neutral comet assay (n=3). Cells were treated with SCR7-cyclized
and SCR7-pyrazine (100 µM and 250 µM) for 24 h and processed for comet assay, followed
by staining with propidium iodidie. DMSO was used as a vehicle control. In case of each
group, three independent images showing multiple cells have been presented. The scale bar
represents 2 µm. F. Scatter plot depicting tail moment of cells processed for neutral comet
assay as analyzed using the CometScore software. A minimum of 50 cells were analyzed in
each batch and the tail moment was plotted for control (grey), 100 µM of SCR7-cyclized and
SCR7-pyrazine (red), 250 µM of SCR7-cyclized and SCR7-pyrazine (black), as a scatter plot
showing mean ± SEM (ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In all cases,
significance was calculated using the Student’s t-test in GraphPad Prism software.
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Figure 5. Accumulation of DNA breaks within the cells upon coadministration of IR
and SCR7-cyclized or SCR7-pyrazine. A. Representative images of HeLa cells showing γ-
H2AX foci upon treatment with IR (1 Gy) in conjunction with (or alone) SCR7-cyclized or
SCR7-pyrazine (100 µM) for 1 h. DMSO was used as vehicle control. γ-H2AX is detected
using Alexa-flour 568 (red), whereas nucleus is stained with DAPI (blue). In each case,
merged image generated using ImageJ is presented. Each experiment was repeated (n=3)
The scale bar represents 0.2 µm. B. Bar graph showing average γ-H2AX foci in each group
depicting mean ± SEM (ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In each
case, a minimum of 100 cells were analyzed for foci formation and the cumulative data was
presented as a bar graph. Significance was calculated using the Student’s t-test in
GraphPad Prism software. C. Representative images showing 53BP1 foci formation in HeLa
cells upon irradiation (1 Gy) and SCR7-cyclized or SCR7-pyrazine treatment (100 µM) for 1
h. 53BP1 foci are detected using FITC (green), nucleus is stained with propidium iodide
(
red). The scale bar represents 0.2 µm. D. Bar graph depicting average number of 53BP1
foci in each cell. Each experiment was repeated (n=3), around 100 cells were analyzed per
batch and the resulting cumulative data was plotted as a bar graph depicting mean ± SEM
(
ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In all cases, significance was
calculated using the Student’s t-test in GraphPad Prism software.
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Figure 6. Assessment of DSB repair kinetics in cells following treatment with SCR7-
cyclized or SCR7-pyrazine in combination with radiation. A-C. Scatter plots showing
number of 53BP1 foci induced post treatment with radiation or inhibitors alone, and in
combination, following a recovery period of 2 h (A), 6 h (B) and 12 h (C). ~100 cells were
analysed in case of each sample (n=3) and plotted as scatter plot showing mean ± SEM (ns:
not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In all cases, significance was
calculated using the Student’s t-test in GraphPad Prism software. D. Line graphs showing
comparative analysis of 53BP1 foci plotted as a time-course post radiation in individual and
combination treatments. Time points chosen for analysis were 1, 2, 6 and 12 h recovery post
IR. Dotted lines represent SCR7-cyclized (red) and SCR7-pyrazine (blue) treatment,
whereas solid lines depict IR alone (black), IR + SCR7-cyclized (red) and SCR7-pyrazine
(
blue). Statistical significance was calculated (Student’s t-test) by comparing number of foci
in IR alone, with that of IR and SCR7-cyclized or SCR7-pyrazine. E. Bar graph depicting fold
change in number of 53BP1 foci over time (difference is compared between 2 h and 12 h).
For each sample, fold decrease in the foci number was calculated at 2 and 12 h time points,
and represented as individual bars for IR alone (black), IR + SCR7-cyclized (red) and IR +
SCR7-pyrazine (blue).
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Figure 7. Effect of SCR7-cyclized and SCR7-pyrazine on cytotoxicity in cancer cell
lines and in a Ligase IV knockout cell line. A-C. Bar graphs representing viable cells as
assessed using trypan blue assay in HeLa (A), MCF7 (B) and Nalm6 (C) upon treatment
with SCR7-cyclized and SCR7-pyrazine (50, 100 and 250 µM) for 48 h. Each experiment
was repeated (n=3) and the percentage of viable cells was plotted as a bar graph showing
mean ± SEM. DMSO was used as a vehicle control. D,E. Bar graph showing comparative
analyses of viable cells in case of Ligase IV knockout cell line, N114 and its wild type, Nalm6
cell line, following treatment with SCR7-cyclized (D) or SCR7-pyrazine (E) for 48 h. In each
case, 50, 100 and 250 µM of inhibitor was used for treatment. DMSO was used as vehicle
control. Each experiment was repeated (n=4) and the percentage viable cells plotted as a
bar graph showing mean ± SEM (ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.0001). In
all cases, significance was calculated using the Student’s t-test in GraphPad Prism software.
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