The function of Poly (ADP-ribose) polymerase 1 in the DNA damage tolerance pathway

  • 孫 延致

Student thesis: Master's Thesis


The DNA damage tolerance (DDT) pathway also known as post-replication repair (PRR) plays an important role in the maintenance of genome integrity Defects in the DDT pathway can cause increasing numbers of stalled DNA replication forks and DNA double strand breaks (DSB) and as a result can cause genomic instability One branch of DDT known as template switching (TS) is regulated by the lysine 63-linked polyubiquitination of PCNA generated by the ubiquitin E2 enzymes UBC13 and MMS2 and the ubiquitin E3 ligases HLTF and SHPRH However the detailed mechanism by which this occurs remains unclear Previously using a coimmunoprecipitation assay we found that HLTF interacts with PARP1 In this study we further identified that HLTF not only interacts with PARP1 but also interacts with BARD1 We revealed that HLTF utilizes HIRAN and the DEXDc domain to interact with PARP1 and utilizes the HIRAN domain to interact with BARD1 Additionally a DNA fiber assay indicated that the depletion of PARP1 reduces the replication track length in response to methyl methanesulfonate (MMS)-induced DNA lesions and simultaneously increases the number of stalled forks Similar results were also shown in the PARP1 null mutant which is generated by the CRISPR-Cas9 system Significantly the PARP1 null mutant exhibits elevated sister chromatid exchanges (SCEs) indicating that an increased number of DSB is generated in the mutant cells The PARP1 null mutant is sensitive to DNA damaging agents such as MMS and cisplatin Furthermore overexpression of the DNA binding domain of PARP1 (PARP1-DBD) can significantly increases SCEs a phenotype similar to the PARP1 null mutant indicating that PARP1-DBD can interfere with the function of endogenous PARP1 Taken together our results suggest that PARP1 is involved in the TS pathway The depletion of PARP1 can result in defective TS and as a result can cause an increasing number of stalled forks and DSBs
Date of Award2018 Jul 20
Original languageEnglish
SupervisorHung-Jiun Liaw (Supervisor)

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