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). Our laboratory focuses on molecular understanding of recombination and DNA repair processes. Using biochemical, structural, microscopic, bioinformatics, single/molecule and genetic approaches we wish to shed light onto mutation avoidance and development of cancer. Candidate can choose from many competitive projects (study of BRCA2 protein, Rothmund-Thomson syndrome helicase, Fanconi anemia proteins, Rad50/Mre11/Xrs2 complex, Srs2 helicase, DNA repair synthesis, Spo11 and meiotic breaks, role of structure specific nucleases etc.) Work in the team of young motivated people at the new campus of Masaryk University, financial bonus awarded every half a year, possibility of study stay at the laboratories of our collaborators.
Our areas of focus:
1.Role of mediator proteins during recombination and repair.
Mutations in the BRCA2 (breast cancer susceptibility 2) gene represent the cause of a significant portion of familial breast cancers and confer an increased risk of ovarian, pancreatic, and prostate cancer (Ford et al., 1998; Wooster et al., 1995). Inactivation of BRCA2 gene function can also result in the cancer prone-syndrome Fanconi anemia (D'Andrea, 2003; Howlett et al., 2002). Despite the prevalence of the BRCA2 gene in cancer etiology, relatively little is known about the molecular function of its encoded product.
The involvement of the BRCA2 protein in HR is evidenced from recent studies showing a physical interaction of this tumor suppressor with Rad51, a key member of the RAD52 epistasis group. Cytological results indicate that BRCA2 is important for the assembly of DNA damage-induced Rad51 nuclear foci (Tarsounas et al., 2003; Yang et al., 2002). BRCA2 has a DNA binding function (Yang et al., 2002) and associates tightly with a small partner protein called DSS1 (Marston et al., 1999), which is also needed for DSB repair by HR (Gudmundsdottir et al., 2004; Kojic et al., 2003).
The available evidence is consistent with the premise that BRCA2 functions as a recombination mediator to deliver Rad51 to ssDNA to facilitate the assembly of the presynaptic filament. We wish to demonstrate and characterize the recombination mediator function of BRCA2 and also to define the molecular basis for this function.
2. Multi-functional role of Srs2 in recombination and DNA repair
The SRS2 (Suppressor of RAD Six) gene appears to be part of such mechanism, as its mutation alleviates the DNA damage sensitivity of cells inactivated for RAD6/RAD18-dependent post-replicative repair (PRR). The accumulated evidences indicate that Srs2 functions as an anti-recombinase to suppress untimely spontaneous recombination events and ensures the channelling of DNA lesions into the Rad6/Rad18-mediated PRR by attenuating the activity of the HR machinery. The SRS2-encoded product belongs to the SF1 helicase family, has ssDNA-dependent ATPase and DNA helicase activities with preference for substrates containing a 3' ssDNA overhang. The helicase activity is enhanced by the inclusion of RPA, possibly through sequestering single-stranded DNA and preventing the reannealing of separated DNA strands. In addition, role of Srs2 has been also implicated in the intra-S-phase checkpoint and in the recovery and adaptation from DNA damage checkpoint-imposed G2/M arrest. The latter function may be related to the ability of Srs2 to remove Rad51 from DNA, as the DNA damaged induced G2/M arrest seen in srs2 mutant cells can be overcome by RAD51 deletion.
In addition to its anti-recombinase function, Srs2 has been implicated to promote DSB repair by Synthesis-Dependent Strand Annealing (SDSA). Srs2 could mediate SDSA in several ways, including removal of Rad51 from ssDNA, thereby preventing the channelling of recombination substrates into pathways that produce crossovers. Alternatively, it seems possible that Srs2 works in DNA synthesis reactions during SDSA through dissociation of the invading DNA strand from the D-loop, and allow annealing of complementary ends. We plan to identify the downstream role of Srs2 as a molecular switch during HR and DNA repair.
Krejčí Lumír, Assoc. prof., M.Sc., Ph.D.
Altmannová Veronika, Ph.D.
Marini Palomeque Maria Victoria, M.Sc., Ph.D.
Mlčoušková Jarmila, Ph.D.
Nikulenkov Fedor, Ph.D.
Špírek Mário, M.A, Ph.D.
Procházková Jana, Ph.D.
Ashraf Raghib, M.Sc.
Štefanovie Barbora, M.Sc.
Sisáková Alexandra, M.Sc. - maternity leave
Hejmalová Lenka, M.Sc.