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dc.contributor.advisorRaghavan, Sathees C
dc.contributor.authorkumari, Nitu
dc.date.accessioned2023-10-04T10:10:50Z
dc.date.available2023-10-04T10:10:50Z
dc.date.submitted2023
dc.identifier.urihttps://etd.iisc.ac.in/handle/2005/6239
dc.description.abstractThe 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 2 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 3 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 4 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
dc.language.isoen_USen_US
dc.relation.ispartofseries;ET00252
dc.rightsI grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertationen_US
dc.subjectAT rich fragile sitesen_US
dc.subjectDNA repairen_US
dc.subjectSSBRen_US
dc.subjectCanceren_US
dc.subject.classificationResearch Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistryen_US
dc.titleA Ligase IV/XRCC4-dependent single strand break repair pathway for the maintenance of A/T-rich regions during DNA replication in mammalsen_US
dc.typeThesisen_US
dc.degree.namePhDen_US
dc.degree.levelDoctoralen_US
dc.degree.grantorIndian Institute of Scienceen_US
dc.degree.disciplineFaculty of Scienceen_US


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