Master's Thesis

Interaction between RECQ4 helicase and Xeroderma Pigmentosum Group A (XPA) protein

Final Thesis 3.68 MB

Author of thesis: Bc. Yuliia Horobets

Acad. year: 2025/2026

Supervisor: Konstantinos Tripsianes, Ph.D.

Reviewer: Ing. Simona Dzurendová, PhD

Abstract:

The investigation of protein-protein interactions is essential for understanding their function in various cellular processes. The RECQ4 protein belongs to the RECQ family of helicases, which are involved in DNA replication and damage repair mechanisms. The xeroderma pigmentosum group A (XPA) protein is an indispensable component of nucleotide excision repair (NER). While an interaction between the two proteins has been previously suggested, the structural details of this interaction remain poorly understood.
The aim of this study was to produce and purify N-terminal fragments
of the RECQ4 (residues 1–150) and XPA (1–97) proteins and analyze their interaction by using nuclear magnetic resonance (NMR) spectroscopy. The recombinant proteins were expressed in Escherichia coli cells and purified using chromatographic methods. The interactions were studied at the atomic level using 2D 1H–15N HSQC titration experiments. Specific changes in the chemical shifts of both proteins were observed, indicating physical interaction. Residue mapping of these changes identified key RECQ4 amino acids involved in the binding. The most prominent perturbations were observed in regions 27–31 and 50–53 of RECQ4. These regions partially overlap with the previously described DNA-binding interface, suggesting a possible regulatory or competitive role for the interaction with XPA. The data obtained provide a molecular insight into the interaction between RECQ4 and XPA and expand our current knowledge of their association.

Keywords:

RECQ4, xeroderma pigmentosum group A, protein-protein interactions, NMR, DNA repair, recombinant protein purification

Date of defence

26.05.2026

Result of the defence

Defended (thesis was successfully defended)

znamkaAznamka

Grading

A

Process of defence

1. Studentka seznámila členy komise s náplní a cílem diplomové práce. 2. Byly přečteny posudky na diplomovou práci. 3. Studentka akceptovala všechny připomínky oponentky a na všechny otázky odpověděla v plné šíři. Diskuse: prof. RNDr. Ivana Márová, CSc. Je xeroderma genově vázané onemocnění? Ve které fázi práce helikázy se váže testovaný protein? Dochází k zastavení funkčnosti helikázy? prof. Ing. Adriána Kovalčík, Ph.D. Používala jste HSQC NMR, mohla byste to objasnit? Na jakém principu tato metoda funguje? doc. Mgr. Pavel Dvořák, Ph.D. Jaký je význam interakcí těchto dvou proteinů pro buňku? Z jakého důvodu zkoumáte tuto interakci? Zmínila jste, že se vyskytuje mutace, jak se tam mutace dostala? Jedná se o cílenou mutaci? Jedná se opravdu o mutaci nebo o polymorfismus? Jak probíhala purifikace proteinů? Jaké další metody by bylo možné použít? Studentka odpověděla na všechny doplňující otázky členů komise, které byly v průběhu diskuse k dané problematice vzneseny. V diskusi studentka prokázala výbornou orientaci v dané problematice. Po diskusi následovalo hodnocení závěrečné práce. Diplomantka prokázala nejen výborné odborné znalosti, ale i schopnost samostatné prezentace dosažených výsledků.

Language of thesis

English

Faculty

Fakulta chemická

Department

Institute of Food Science and Biotechnology

Study programme

Chemistry for Medical Application (NPCP_CHMA)

Specialization

Chemistry of Bioactive Substances (CHBL)

Composition of Committee

prof. RNDr. Ivana Márová, CSc. (předseda)
doc. Ing. Petr Sedláček, Ph.D. (místopředseda)
doc. Ing. Pavel Diviš, Ph.D. (člen)
prof. Ing. Adriána Kovalčík, Ph.D. (člen)
doc. Ing. Eva Vítová, Ph.D. (člen)
doc. Mgr. Pavel Dvořák, Ph.D. (člen)

Supervisor’s report
Konstantinos Tripsianes, Ph.D.

Yuliia Horobets studied the interaction between RecQ4 and XPA proteins. She is a fast learner and after short training she was able to work indepedently in the Biochemistry lab. Not only she manged to purify both proteins, she also troubleshot several steps and established reproducible purification protocols. She was succesfull in monitoring the interaction by NMR and draw some interesting conclusions for follow-up studies. She presented her findings 3-4 times in our group seminars in a very clear and professional manner. At the end, she presented a layout of her thesis and drafted most parts by herself. Overall she proved to be a valuable member that communicated effectively within the group, posted research questions, worked hard in reaching her goals (many times she had to commute from VUT to CEITEC), had a good overview of literature, and presented her findings in a concise way.
Evaluation criteria Grade
Splnění požadavků zadání A
Studium literatury a její zpracování A
Využití poznatků z literatury B
Kvalita zpracování výsledků A
Interpretace výsledků, jejich diskuse A
Závěry práce a jejich formulace A
Využívání konzultací při řešení práce A
Celkový přístup k řešení úkolů A

Grade proposed by supervisor: A

Reviewer’s report
Ing. Simona Dzurendová, PhD

Yuliia Horoberts investigates the molecular details of the interaction between the N-terminal domains of two key proteins closely involved in DNA replication and repair pathways: RECQ4 helicase and the Xeroderma Pigmentosum Group A (XPA) protein. The thesis follows a well-structured and logical format, provides a comprehensive literature review, and all defined thesis objectives were completely fulfilled. In the experimental section, the author successfully mastered challenging methodological workflows involving the recombinant expression and multi-step purification of both protein fragments (RECQ4 1–150 and XPA 1–97), including the correction of an unwanted point mutation via site-directed mutagenesis within the RECQ4 construct in the pETM-11 plasmid. The purification protocols utilized advanced chromatographic techniques (IMAC, IEX, and SEC), and for the XPA protein, a boiling step was integrated to eliminate host-cell impurities. The interaction of XPA and RECQ4 was evaluated using 1H–15N HSQC NMR spectroscopy, where chemical shift perturbations in both proteins revealed interaction between the two N-terminal regions. Overall, the thesis of Bc. Yuliia Horobets is written in high-quality English, presents original and robust data, and I fully recommend it for defense with the final grade A.
Evaluation criteria Grade
Splnění požadavků zadání A
Logické členění práce A
Kvalita zpracování výsledků A
Interpretace výsledků, jejich diskuse A
Využití literatury a její citace A
Úroveň jazykového zpracování A
Formální úroveň práce – celkový dojem A
Závěry práce a jejich formulace A
Topics for thesis defence:
  1. Počas analýzy čistoty RECQ4 1–150 pomocou SDS-PAGE ste zaznamenali indikácie čiastočnej degradácie proteínu. Aké činidlá sú pridávané v priebehu purifikácie pre zabránenie degradácie? Aké faktory musíme zohľadniť pre zabránenie agregácie fragmentov?
  2. Pri charakterizácii fragmentov XPA 1–97 a RECQ4 1–150 ste využívali 2D 1H–15N HSQC NMR spektroskopiu. Ako sa v tomto type spektra na prvý pohľad vizuálne prejaví, že je analyzovaný proteín (alebo jeho časť) priestorovo zbalený (folded), v porovnaní so stavom, kedy je proteín neštruktúrovaný či denaturovaný (unfolded)?

Grade proposed by reviewer: A

Responsibility: Mgr. et Mgr. Hana Odstrčilová