Unit:
Κατεύθυνση Ιατρική Φυσική-ΑκτινοφυσικήLibrary of the School of Health Sciences
Author:
Vasileiou Christina
Supervisors info:
Αναστάσιος Σιούντας, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΑΠΘ, Επιβλέπων
Αλέξανδρος Γεωργακίλας, Αναπληρωτής Καθηγητής, Σχολή Εφαρμοσμένων Μαθηματικών και Φυσικών Επιστημών, ΕΜΠ
Γεωργία Τερζούδη, Ερευνήτρια Α΄, ΕΚΕΦΕ "Δημόκριτος"
Original Title:
Characterization of kinases involved in DNA damage response checkpoints after low and high doses of low-LET irradiation and detection of structural DNA variations after induction of complex DNA damage using Nanopore sequencing.
Translated title:
Characterization of kinases involved in DNA damage response checkpoints after low and high doses of low-LET irradiation and detection of structural DNA variations after induction of complex DNA damage using Nanopore sequencing.
Summary:
Cancer treatments such as radiotherapy and chemotherapy mainly act by inducing severe (complex) damage on DNA of cancer cells. In presence of DNA damage, cells implement their multifaceted DNA Damage Response (DDR) system to process the damage and repair it. Checkpoint control plays a major role during this response, therefore targeted inhibition of the aforesaid can lead to increased cell lethality and potentially to more efficient treatments (Bartek and Lukas, 2001; Iliakis et al., 2003; Jackson and Zhou, 2004).
Here, using data generated by cells exposed to low and high doses of low LET ionizing radiation that introduce a low number of double strand breaks (DSBs) in the genome, we cross validate the regulation of the G2 checkpoint mainly by ATR and ATM (results first published by (Mladenov et al., 2019)). We also provide evidence that these kinases interphase with cell cycle machinery mainly through Chk1 kinase. At high doses, we present preliminary results of the dominant role of Chk1 in checkpoint control.
High number of DSBs is known to cause even more severe cell damage (Khanna and Jackson, 2001; van Gent et al., 2001). One of the exogenous agents inducing multiple DSBs is the high Linear Energy Transfer (LET) ionizing radiation. One of the characteristics of high LET irradiation is the introduction of highly complex lesions. This complexity is traditionally thought to be the combination of lower level lesions like one stranded lesions, base changes or losses, SSBs, DSBs etc. or a combination of the previous mentioned types of damage. The combination of low level lesions with DSBs poses major threats to genome integrity according to literature (Campa et al., 2005; Gupta et al., 2009; Seluanov et al., 2010). Here we have used a model based on the hypothesis that increased complexity of DNA damage is manifested through the colocalization or combination of an increased number of DSBs in a relative small stretch of sequence (Iliakis et al., 2018; Mladenova et al., 2016).
The cellular clones used for this part of the analysis are CHO and human clones that were created with the aim to model DSB-clusters. The I-SceI recognition sequence was inserted into cell lines using molecular biology techniques (Jasin, 1996) and clones as needed were selected and characterized. I-SceI recognition sequences introduced in a genome can then be cut, to generate from simple DSBs up to more complex DSB clusters. Constructs carrying different combinations of I-SceI sites were engineered at specific distances and orientations, subsequent transient expression of I-SceI induces the relevant number of DSBs in predetermined loci. Therefore these clones provide the opportunity to measure the effect of several DSBs within a relatively small part of a sequence, excluding postulations about other variables and types of DNA damage that arise when high LET IR is used. A characterization of these clones was performed using long-read sequencing with MinION platform by ONT. Localization and identification of the I-SceI sites is presented on both human and CHO clones and a novel model is proposed to study induced structural variations. Results reveal significantly more integrations sites in the clones tested in comparison to other techniques. In addition, compromised DSB repair as well as increased translocation formation were evident.
Main subject category:
Health Sciences
Keywords:
Ionizing radiation, Complex DNA damage, DNA damage response, Low doses, Nanopore sequencing, MinION, ONT, Bioinformatics, High LET, Low LET, ATM, ATR, Chk1, Chk2, G2 checkpoint, Cell cycle
