| dc.description.abstract | The DNA in our cells is under constant threat from various exogenous and
endogenous sources. To ensure error free and faithful transmission of hereditary material,
human cells employ several DNA repair pathways. Mammalian cells utilize multiple DNA
repair mechanisms: base excision repair (BER), mismatch repair (MMR), nucleotide excision
repair (NER), and double-strand break repair, which includes homologous recombination
(HR), nonhomologous end joining (NHEJ) and recently discovered microhomology mediated
end joining (MMEJ) or Alt-NHEJ. While NHEJ entails the direct end-to-end joining of the
broken ends, HR requires genetic information from the homologous DNA sequence from the
partner chromosome for repair.
Often cancer progression has been associated with alterations in DNA repair genes
caused by mutations. Different NHEJ repair proteins have been shown to be upregulated in
several cancers, such as breast cancer, gastric cancer, oral squamous cell carcinoma,
oesophageal cancer, and lung cancer. Ligase IV and XRCC4 play a key role in the joining of
DSBs during NHEJ and displayed increased expression in a pan-cancer manner in
comparison to healthy cells.
In the first part of the current study, we investigated the sensitivity of LIG4 mutant
cells toward different FDA-approved drugs. For that, we have generated 6 different mutants
of LIG4 in cervical cancer and normal kidney epithelial cell lines using CRISPR-Cas9
mediated genome editing. LIG4 mutant cells exhibited compromised cell viability, reduced
NHEJ, and accumulation of DSBs due to slow repair kinetics. Interestingly, LIG4 mutant cells
showed increased MMEJ-mediated repair and an increase in sensitivity toward clinically
relevant drugs. Finally, we demonstrate that when combined with IR, LIG4 mutant cervical
cancer cells are highly sensitive to FDA-approved drugs.
Single-strand breaks (SSBs) can arise either directly e.g., from the attack of
deoxyribose by free radicals such as reactive oxygen species (ROS) or indirectly via
enzymatic cleavage of the phosphodiester backbone e.g., as normal intermediates of DNA
base excision repair (BER). The repair of direct and indirect SSBs has collectively been
termed single-strand break repair (SSBR), primarily because the same group of proteins
appears to repair both types of breaks. SSBR can be divided into four basic steps. Most
indirect SSBs are created during BER by AP endo-nuclease (APE1), and are then “handed”
to the next enzyme in the BER process in a molecular relay. SSBR starts with DNA damage
binding by Poly (ADP-ribose) polymerase (PARP) followed by DNA end processing. The
third step is DNA gap filling primarily by DNA pol β, followed by DNA ligation by DNA Ligase
III/XRCC1. To date, there has been a widespread assumption that DNA repair inside a cell is
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being carried out irrespective of the DNA sequence. Surprisingly, in our laboratory, it was
observed that cell-free extracts prepared from various mammalian tissues could join single stranded oligomeric DNA only when thymine DNA sequences were present (Srivastava,
2013). In the present study, we have investigated the molecular mechanism of this
interesting observation. Various lines of experimentation showed that among the different
DNA ligases (Ligase I, Ligase III/XRCC1, and Ligase IV/XRCC4), only DNA Ligase
IV/XRCC4 complex could catalyse the joining of ssDNA harbouring poly Ts. This observation
is very fascinating, considering Ligase IV/XRCC4 is only known to ligate DNA double-strand
breaks during nonhomologous end joining. EMSA studies showed that Ligase IV/XRCC4
complex could bind to ssDNA Poly (T)35, however the joining of single-stranded poly T
requires Ligase IV/XRCC4 complex and cannot be catalyzed by either of the protein alone.
Biolayer interferometry (BLI) studies showed that full-length Ligase IV is required for the
stable binding of the protein to the single-stranded DNA of poly Ts. DNA bubble substrate
mimicking the DNA replication and transcription intermediates exhibited efficient joining
when poly Ts were present at the bubble. Although Ligase IV/XRCC4 could join these
substrates when poly Ts were present, the efficacy of the joining was significantly improved
when incubated with rat testicular extract. Besides, the sequence specificity remained
consistent even when investigated in double-stranded DNA context.
Importantly, single-stranded DNA containing poly thymine in dsDNA context showed
joining by rat testicular extract but not by purified Ligase IV/XRCC4 complex, suggesting
additional factors were required. The optimum joining of single-stranded DNA containing
poly thymine in dsDNA context occurred at 25ºC with 2 µg of extract in 30 min of incubation.
The optimum concentration of MgCl2 required for joining was 7.5 mM and that of ATP was
0.5 mM.
Reconstitution experiments using proteins associated with NHEJ revealed the joining
of double-stranded DNA but not those containing ssDNA with poly thymine in dsDNA
context, suggesting that additional proteins are involved in the joining of such single
stranded DNA breaks possessing long poly T sequences. To identify the additional proteins
involved in this novel repair mode, rat testicular extract was fractionated and evaluated.
Interestingly, we observed that fractions required for the joining of NHEJ DNA substrates
and ssDNA containing poly thymine in dsDNA context substrate were different, indicating the
requirement of a different set of DNA repair proteins. Pull down using biotinylated ssDNA
containing poly thymine in dsDNA context substrate after incubation with specific fractions
(with maximum joining activity) followed by mass spectrometry analysis showed different
proteins from various repair pathways including HR, NHEJ, NER, and BER. The binding of
RAD50, Ligase IV/XRCC4, RPA, CtIP, and MRE11 with ssDNA containing poly thymine in
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dsDNA context were confirmed by EMSA, followed by western blot analysis.
Immunoprecipitation of Ligase IV followed by western analysis with supernatant fraction
showed a reduction in the level of RPA, MRE11, RAD50, Pol μ, and CtIP protein, confirming
the presence of these proteins in the complex. Further, in vitro pull-down with purified Ligase
IV/XRCC4 showed that RPA, CtIP, and RAD50 can directly interact with Ligase IV. Joining
of ssDNA containing poly thymine in dsDNA context with immunoprecipitated extract of RPA,
MRE11, RAD50, Pol μ and CtIP showed a significant reduction in the joining, suggesting the
involvement of these proteins in the thymine dependent joining of SSBs.
The previous report indicates that poly (dA:dT) tracts could be preferential sites of
polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs), late replicating common fragile sites (CFSs), and at the rDNA replication fork barrier (Tubbs et
al., Cell, 2018). To understand the role of Ligase IV at the stalled fork, DNA fiber assay was
carried out in the presence of hydroxyurea (HU), which is known to induce SSBs and, thus
fork stalling. Our results showed a significant increase in fork stalling when Ligase IV was
knocked out in cells through CRISPR-Cas9 mediated gene editing, suggesting the role of
Ligase IV in replication fork restart. Interestingly, there was no effect on replication fork
restart upon KU70 knockdown suggesting the role of Ligase IV in replication fork restart,
independent of NHEJ. Further, we found that Ligase IV/XRCC4 is present at the replication
site by employing nascent DNA pull-down assay and mass spectrometry studies. Besides,
accumulation of ssDNA upon HU treatment was detected by native BrdU incorporation
assay in Ligase IV knock out (KO) cells. Similarly, a significant increase in RPA foci in
Ligase IV KO cells following HU treatment demonstrates the accumulation of single-stranded
DNA in KO cells. Importantly, ChIP sequencing analysis revealed that Ligase IV and RPA
can colocalize at poly dT/dA sequences, suggesting the binding of Ligase IV at single stranded poly dT/dA regions inside the genome. Taken together, our results suggest that
Ligase IV is involved in repairing SSBs generated at the stalled replication fork.
The ChIP assay using anti-Ligase IV showed hydroxyurea treatment dependent
binding of Ligase IV at several AT-rich fragile sites. Ligase IV KO cells showed no binding of
Ligase IV to AT-rich fragile sites, suggesting that binding was specific to Ligase IV. ChIP
sequencing with Ligase IV antibody in presence and absence of HU showed enrichment of
Ligase IV binding peaks at AT-rich regions. These results indicates that Ligase IV is involved
in the maintenance of common AT-rich fragile sites associated with several pathological
conditions.
Thus, the results of the study provide new insights into a previously uncharacterized
single-stranded DNA break repair pathway, which is dependent on DNA Ligase IV. Our
study suggests that single-strand breaks in thymine rich DNA generated during DNA
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replication can lead to fork arrest, and under these conditions, replication restart is
dependent on the function of Ligase IV in NHEJ independent manner. We revealed that
RPA, CtIP, RAD50, MRE11, and Ligase IV are present in the complex, and the function of
Ligase IV in fork restart appears to be because of their mutual interactions. Overall, we
provide new evidence for NHEJ-independent functions of DNA Ligase IV in novel single stranded DNA break repair pathways, which might explain the crucial nature of this protein in
the survival of organisms. | en_US |
