DNA double-strand breaks (DSB) are among the most lethal forms of DNA damage. DSBs are induced by a large number of endogenous and exogenous agents, as genotoxic chemicals and ionizing radiation, or due to replication of damaged DNA as well as collapse of a replication fork. DSB can either be properly repaired, restoring genomic integrity, or miss-repaired resulting in cell death, genomic instability, and cancer. Our group focuses on mechanisms of DSB repair using multi-disciplinary approach (biochemical, structural, computational, genetic, and single-molecule).
Research
The integrity of DNA continually resists the presence of physical and chemical carcinogens in our environment. In addition to exogenous agents, DNA undergoes spontaneous decay, including replication errors, oxidative and other damages which arise from common metabolic processes. The repair of damaged DNA is vital for the maintenance of genome integrity, and as a result, all organisms have evolved a wide variety of DNA repair pathways that can restore DNA structure and its genetic information.
The main objective of our research is to decipher the intrinsic functions of homologous recombination (HR) which has a dual role in the maintenance of genome stability. First, it promotes the faithful repair of DNA double-strand breaks (DSBs) belonging among of the most lethal forms of DNA damage. Moreover, HR is responsible for the creation of genetic variability during meiosis by directing the formation of reciprocal crossovers that result in random combinations of alleles and traits. Changes in the execution and regulation of recombination are linked to human infertility, miscarriage and genetic diseases, particularly cancer thus emphasizing the importance of better understanding the mechanism and regulation of this pathway.
To achieve our goals, we utilize a wide range of different methods from biochemistry, molecular biology, genetics, structural biology, and biophysics that are well established in our lab. Since we believe that interdisciplinary approach is needed to fully understand the fundamental biological processes, we also collaborate with numerous specialists.
Currently, our main research focus comprises these topics:
Inhibitors of nucleases
Nucleases play an essential role in various DNA repair pathways from processing the damaged nucleotide and extensive resection of the DNA ends to resolution of various DNA intermediates. For these reasons, nucleases comprise an integral part of many repair pathways and their inactivation results in the accumulation of DNA damage, consequentially leading to genomic instability and cancer. Indeed, overexpression of nucleases often results in cancer cells being resistant to chemo- or radiation therapy (1).
Therefore, inhibiting nucleases will be beneficial to the sensitization of cancer cells to various anticancer therapies currently in use. In addition, since nucleases are downstream repair factors and present the possibility for inhibiting their enzymatic activities more specifically, their inhibitors are expected to be extremely useful in personalized medicine using the synthetic lethal approach (2).
Implementing a very interdisciplinary approach, namely combination of organic synthesis/medicinal chemistry, biochemistry and molecular and cell biology we wish to develop potent and selective inhibitors of nucleases for possible therapeutic use and further characterize their role in maintenance of genome integrity.
Table – DDR proteins as therapeutic targets
RAD51 and its paralogs
RAD51 protein functions as central mitotic recombinase forming helical nucleoprotein filament capable of homology search and strand invasion. Formation of RAD51 filament represents a key regulatory step during early phases of HR. RAD51 nucleation and filament growth is controlled by a variety of recombination modulators such as factors promoting filament formation and stimulating its activity, known as recombination mediators or proteins dismantling RAD51 presynaptic filament thereby preventing harmful recombination events, referred to as antirecombinases (3).
RAD51 filament is able to search for and invade homologous duplex DNA, and also protect the DNA from degradation at damaged replication forks, but relies on numerous co-factors (BRCA2, RAD51 paralogs, RAD51AP1) to drive the HR reaction to completion. However, a mechanistic understanding of how these co-factors act is unclear and remains a significant challenge to the field (3,4).
Through the integration of biochemical, real-time kinetic measurements, microscale thermophoresis, conformational dynamics, and validation in biological systems we would like to describe the mechanisms by which HR co-factors impact on the HR reaction and replication. Deciphering these various regulations could hold the key to understanding the cancer-prone nature of mammalian cells, molecular mechanism of cancerogenesis and possibly unravel novel RAD51 filament-targeting strategies suitable for treatment of therapy-resistant tumours.
Regulation of RAD51 presynaptic filament formation. RAD51 filament formation and stability is controlled by various factors. Recombination mediators such as BRCA2 promote presynaptic filament assembly, meanwhile antirecombinases can dismantle RAD51 filament thus suppressing HR. RAD51 paralogs were shown to regulate RAD51 filament formation, however the mechanism of their action remains unknown.
Sumoylation
HR is absolutely crucial to the genomic stability of the cell, an untimely, uncontrolled or not proper recombination can be very harmful, indicating a requirement for a tight regulation. In recent years, increasing evidence indicate that SUMO (small ubiquitin-related modifier) plays a critical role during post-translational modification in HR regulation. Sumoylation has been shown to regulate many fundamental cellular pathways such as gene transcription, intracellular transport, DNA replication and repair, chromosome segregation, basic metabolism, ion and protein transport, cellular senescence and ageing (5).
Though the importance of sumoylation in the HR regulation is substantial, its exact role remains often elusive. However, several obstacles impede an understanding of the role of sumoylation in regulation of protein function, including identification of modified sites and their possible redundancy, identification of the downstream SUMO-interacting partners, analysis of sumo-deficient alleles as well as permanent sumo-fusion of target proteins that can only partially mimic the effect of sumoylation.
Our aim is to understand the complex effects of SUMO modification on the activities, localizations and interactions of HR proteins and to uncover the molecular mechanism and biological significance of sumoylation in HR. Moreover, understanding the role of sumoylation in various steps of DSB repair and its molecular characterization could also present tool for small molecule intervention with possible therapeutic applications.
The double-strand break repair pathways in S. cerevisiae. After DNA damage, DSBs can be either resected to generate 3’ ssDNA tails and directly ligated by non-homologous end joining (NHEJ) (I) or processed by homologous recombination (HR) (II). In HR, resection of a DSB is followed by formation of a Rad51 presynaptic filament invading into the homologous strand to form a D-loop structure. The invading strand is then extended by DNA synthesis. The resulting extended D-loop could then be processed by one of three alternative mechanisms: synthesis-dependent strand annealing (SDSA) (A); double-strand break repair (DSBR) (B); or break-induced replication (BIR) (C); Proteins involved in DSB repair that undergo sumoylation are depicted. An alternative pathway–single strand annealing (SSA)–can be used for DSBs occurring between repeated DNA sequences (D) (5).
References
1. Bartosova Z, Krejci L. Nucleases in homologous recombination as targets for cancer therapy. FEBS Lett. 2014;588(15):2446-56.
2. Samadder P, Aithal R, Belan O, Krejci L. Cancer TARGETases: DSB repair as a pharmacological target. Pharmacol Ther. 2016;161:111-31.
3. Krejci L, Altmannova V, Spirek M, Zhao X. Homologous recombination and its regulation. Nucleic Acids Res. 2012;40(13):5795–818.
4. Taylor MRG, Spirek M, Chaurasiya KR, Ward JD, Carzaniga R, Yu X, Egelman EH, Collinson LM, Rueda D, Krejci L, Boulton SJ. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination. Cell. 2015;162(2):271–86.
5. Altmannova V, Kolesar P, Krejci L. SUMO Wrestles with Recombination. Biomolecules. 2012;2(3):350-75.
