| الملخص: | <p>Genome maintenance is a vital biological process for humans. DNA interstrand crosslink (ICL) is a type of DNA damage that can fail DNA replication and transcription. The Fanconi Anemia pathway (the FA pathway) is the key process for repairing ICLs. UHRF1 acts as the sensor to recognize ICLs, which then recruits Fanconi Anemia (FA) proteins and FA-associated proteins, including FANCD2. The mono-ubiquitination of FANCD2 is essential for activating the downstream FA pathway. This ubiquitination process is regulated by the CK2/PP2A axis and the ATR-mediated phosphorylation of FANCD2. Besides, UHRF1 is also known to be the partner of DNMT1 for the protection of the epigenome. However, three questions about these known processes require further exploration.</p>
<p>The first gap in knowledge relates to the structure of full-length UHRF1, and how UHRF1 recognizes ICLs within chromatin. To address this, I used UHRF1 to form a complex with a nucleosome containing an ICL at the DNA tail. After confirmation through Western Blot (WB), native gel electrophoresis with ethidium bromide (EB) staining, gel filtration, and negative staining, the complex of UHRF1 and nucleosome can be prepared stably in vitro. Currently, the complex is undergoing cryo-EM screening to elucidate its structure and understand the mechanism by which UHRF1 recognizes ICLs within chromatin.</p>
<p>The second question is the order of the CK2/PP2A axis and ATR in regulating FANCD2. To solve this problem, we purified wild-type (WT) FANCD2, as well as phospho-mimicking and phospho-deficient mutations of FANCD2 from HeLa cells. These mutated residues are the targets of CK2 and ATR. Subsequently, these protein samples will be analysed by liquid chromatography–tandem mass spectrometry (LC-MS/MS).</p>
<p>The last question is whether DNMT1 also mediates the response to ICLs. Our findings suggest that DNMT1 is recruited to the DNA damage region during excessive ICL formation. Also, knocking down DNMT1 rescues the hypersensitivity of HeLa cells to ICLs induced by FANCD2 depletion. From these results, we speculate that DNMT1 may mediate severe DNA lesion-induced cellular apoptosis. However, the specific mechanisms by which DNMT1 affects programmed cell death, and whether UHRF1 also participates in this process, need to be investigated. We hypothesize that DNMT1-induced DNA hypermethylation may play a role in this process, since hypermethylation is associated with cellular apoptosis. Further experiments will be designed to clarify the details of this process.</p>
<p>Taken together, my work is aimed at uncovering the roles of proteins in the FA pathway which mediates ICLs repair.</p>
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