Federica Marini
e-mail: federica.marini AT unimi.it
affiliation: Università di Milano
research area(s): Molecular Biology, Cancer Biology
Course:
Biomolecular Sciences
University/Istitution: Università di Milano
University/Istitution: Università di Milano
Federica Marini had her degree in Biological Sciences (110/110 summa cum laude) at the University of Milano, Italy. In 1997 she obtained her PhD in Genetics at the same University. From 1998 to 2001 she worked as a Postdoctoral fellow in R. D. Wood"s laboratory, Cancer Research UK. From 2001 to 2003 she worked as a Research Associate at the University of Pittsburgh Cancer Institute, USA. Since 2003 she has been working at the Dip. di Scienze Biomolecolari e Biotecnologie, University of Milan. In 2006 she obtained a permanent position as a Researcher at the University of Milano (BIO11, Molecular Biology). She is the author of several publications on well-recognized peer-reviewed international journals. Her research has been and is funded by several national and international agencies and she has been invited to give seminars in several national and international research institutes and universities.
The scientific activity of Federica Marini has been always focused on the study of the strategies that the cell follows when its DNA is damaged. During her PhD thesis, the object of her research was the mechanisms of genome surveillance, also called DNA damage checkpoints, in S. cerevisiae. In R. D. Wood"s laboratory, her main focus has been DNA repair and cancer research in connection to some inherited human syndromes, characterized by DNA repair defects and genome instability. In particular, F. Marini"s main interest has been the repair of Interstrand Cross-Links (ICLs), a very toxic type of DNA damage caused by many chemotherapeutic agents. She isolated and characterized a novel DNA helicase and DNA polymerases that are involved in ICL repair. In collaboration with Simon Boulton, Cancer Research, UK, she investigated their cellular functions using C. elegans as a model system. She discovered an important function of the helicase HELQ in the late steps of homologous recombination. In parallel she has established a line of research on the connections between DNA repair and checkpoint activation, working on inherited human diseases, characterized by genome instability. Her recent work is focusing on helicase and translocases involved in DNA recombination and cancer predisposition.
1. Marini F.*, Nardo T., Giannattasio M., Minuzzo M., Stefanini M., Plevani P., and Muzi Falconi M. (2006) DNA Nucleotide Excision Repair-Dependent Signaling to Checkpoint Activation. Proceedings of the National Academy of Sciences of the United States of America 103;17325-17330.
2. Muzzini D.M., Plevani P., Boulton S.J., Cassata G. and Marini F.* (2008) C.elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways. DNA repair 7; 941-950.
3. Ward J.D., Muzzini D.M., Petalcorin M.IR, Perez E.M., Martin J.S., Plevani P., Cassata G., Marini F.*, Boulton S.J. (2010) Overlapping mechanisms promote post-synaptic RAD-51 filament disassembly during meiotic double-strand break repair. Molecular Cell 37; 259-272.
4. Tumini E., Plevani P., Muzi Falconi M. and Marini F. * (2011) Physical and functional crosstalk between Fanconi anemia core components and the GINS replication complex. DNA repair 10;149-158.
5. Sertic S., Pizzi S., Cloney R., Lehmann A.R., Marini F., Plevani P. and Muzi-Falconi M. (2011) Human Exonuclease 1 connects NER processing with checkpoint activation in response to UV irradiation. Proceedings of the National Academy of Sciences of the United States of America (in press).
2. Muzzini D.M., Plevani P., Boulton S.J., Cassata G. and Marini F.* (2008) C.elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways. DNA repair 7; 941-950.
3. Ward J.D., Muzzini D.M., Petalcorin M.IR, Perez E.M., Martin J.S., Plevani P., Cassata G., Marini F.*, Boulton S.J. (2010) Overlapping mechanisms promote post-synaptic RAD-51 filament disassembly during meiotic double-strand break repair. Molecular Cell 37; 259-272.
4. Tumini E., Plevani P., Muzi Falconi M. and Marini F. * (2011) Physical and functional crosstalk between Fanconi anemia core components and the GINS replication complex. DNA repair 10;149-158.
5. Sertic S., Pizzi S., Cloney R., Lehmann A.R., Marini F., Plevani P. and Muzi-Falconi M. (2011) Human Exonuclease 1 connects NER processing with checkpoint activation in response to UV irradiation. Proceedings of the National Academy of Sciences of the United States of America (in press).
Project Title:
Helicase and translocases involved in the maintenance of genome stability and cancer predisposition
Abstract
BACKGROUND: Homologous recombination (HR) is a central pathway to maintain genomic stability. RAD54 and RAD54B are two paralogs, which belong to the Swi2/Snf2 family of ATP hydrolysis-dependent chromatin remodelers and traslocases. In vitro, their DNA translocase activity can modify DNA topology, enhance D-loop formation by the Rad51 recombinase and accelerate the rate at which DNA strands are exchanged during the HR reaction. Remarkably, RAD54 and RAD54B also catalyze the removal of Rad51 from double-stranded DNA filaments. We recently showed that the DNA helicase HELQ is also able to disassemble RAD51 from dsDNA.
AIMS OF THE PROJECT: The research project aim to explore the interplay between HELQ, RAD54 and RAD54B during HR in human cell lines. The efforts of the candidate PhD student will be directed: i) to study whether the three proteins physically interact by co-immunoprecipitation and to investigate possible post-translational regulatory modifications of HELQ, RAD54 and RAD54B. ii) to set up a biochemical screening to identify novel RAD54B interactors to better understand RAD54B cellular functions. iii) Finally, he/she will investigate the interplay between HELQ, RAD54 and RAD54B at a unique double-strand break (DSB) in vivo by chromatin immunoprecipitation and immunofluorescence.
The expected results should contribute to shed some light on the role of HELQ, RAD54 and RAD54B in HR and how they regulate RAD51 activity. A fine-tuning of HR is pivotal to genome stability. In fact, mutations in both RAD54 and RAD54B are found in primary cancers and Rad51 is over-expressed in p53-negative tumor cells, leading to increased resistance to drugs used in chemotherapies.
Experimental approach and methodology
TASK 1: a) Identification of the physical connections between HELQ, RAD54 and RAD54B.
HELQ, RAD54 and RAD54B are all able to disassemble dsDNA-RAD51 filaments in vitro. Thus, it is important to study whether HELQ, RAD54 and RAD54B physically interact. The candidate will perform co-immunoprecipitation experiments both in normal growth conditions, following DNA damage and in different cell cycle phases. He/she will analyze by gel filtration whether they belong to the same complex.
b) Post-translational modification of HELQ, RAD54 and RAD54B.
It is known that the main DNA damage checkpoint kinases (ATM, ATR, CHK1, CHK2) phosphorylate and activate many factors in the presence of DSB, among which also several proteins involved in HR.
The aim of this task is to discover whether HELQ, RAD54 and/or RAD54B are post-translationally modified following DSB formation and/or in any phase of the cell cycle.
Interestingly, our preliminary results indicate that RAD54B is phosphorylated in M phase by cyclin-dependent kinases. The candidate will investigate the functional meaning of this post-translational modification.
He/she should also consider other possible post-translational modifications like ubiquitylation and sumoylation.
Milestones: co-immunoprecipitations: 3-6 months; gel filtration: 3 months. Study of post-translational modifications: 3-6 months.
TASK 2: Identification of RAD54B interactors in the presence of DSB lesions.
In order to identify RAD54B interactors, we already started a biochemical approach. We cloned RAD54B in frame with the GST sequence. The peptides are very well expressed and we are setting up the conditions to prepare affinity columns with which the candidate will pull-down putative protein interactors that will then be identified by tandem mass spectrometry (MS/MS), in collaboration with Prof. Corrado Santocanale (NUI Galway). Depending upon the results the candidate will then focus his/her efforts on understanding the functional meaning of the interaction between RAD54B and few interesting putative binding proteins.
Milestones: Biochemical screening: 12 months. Characterization of identified factors: 24 months.
TASK 3: Investigation of the interplay between HELQ, RAD54 and RAD54B at a unique double-strand break in vivo.
In order to study HELQ, RAD54 and RAD54B involvement in HR, the candidate will monitor a single DNA repair event by immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. U2OS-DRGFP cell line that bears a modified GFP gene in which an I-SceI restriction site has been engineered will be used (from Maria Jasin). The candidate PhD student will monitor whether RAD54, RAD54B and HELQ form a single focus, co-localizing with RAD51 and γ-H2AX.
RAD54B, RAD54 and HELQ could bind very close to the break or, being involved in the displacement of RAD51 from dsDNA, they could bind far from the break site. One attractive strategy is to examine the assembly of RAD54B, RAD54 and HELQ on a DSB using ChIP with primer pairs specific to regions of interest proximal or progressively far away from the DSB created by I-SceI.
Milestones: localization at a single DSB: 3-6 months; ChIP analysis: 12 months.
BACKGROUND: Homologous recombination (HR) is a central pathway to maintain genomic stability. RAD54 and RAD54B are two paralogs, which belong to the Swi2/Snf2 family of ATP hydrolysis-dependent chromatin remodelers and traslocases. In vitro, their DNA translocase activity can modify DNA topology, enhance D-loop formation by the Rad51 recombinase and accelerate the rate at which DNA strands are exchanged during the HR reaction. Remarkably, RAD54 and RAD54B also catalyze the removal of Rad51 from double-stranded DNA filaments. We recently showed that the DNA helicase HELQ is also able to disassemble RAD51 from dsDNA.
AIMS OF THE PROJECT: The research project aim to explore the interplay between HELQ, RAD54 and RAD54B during HR in human cell lines. The efforts of the candidate PhD student will be directed: i) to study whether the three proteins physically interact by co-immunoprecipitation and to investigate possible post-translational regulatory modifications of HELQ, RAD54 and RAD54B. ii) to set up a biochemical screening to identify novel RAD54B interactors to better understand RAD54B cellular functions. iii) Finally, he/she will investigate the interplay between HELQ, RAD54 and RAD54B at a unique double-strand break (DSB) in vivo by chromatin immunoprecipitation and immunofluorescence.
The expected results should contribute to shed some light on the role of HELQ, RAD54 and RAD54B in HR and how they regulate RAD51 activity. A fine-tuning of HR is pivotal to genome stability. In fact, mutations in both RAD54 and RAD54B are found in primary cancers and Rad51 is over-expressed in p53-negative tumor cells, leading to increased resistance to drugs used in chemotherapies.
Experimental approach and methodology
TASK 1: a) Identification of the physical connections between HELQ, RAD54 and RAD54B.
HELQ, RAD54 and RAD54B are all able to disassemble dsDNA-RAD51 filaments in vitro. Thus, it is important to study whether HELQ, RAD54 and RAD54B physically interact. The candidate will perform co-immunoprecipitation experiments both in normal growth conditions, following DNA damage and in different cell cycle phases. He/she will analyze by gel filtration whether they belong to the same complex.
b) Post-translational modification of HELQ, RAD54 and RAD54B.
It is known that the main DNA damage checkpoint kinases (ATM, ATR, CHK1, CHK2) phosphorylate and activate many factors in the presence of DSB, among which also several proteins involved in HR.
The aim of this task is to discover whether HELQ, RAD54 and/or RAD54B are post-translationally modified following DSB formation and/or in any phase of the cell cycle.
Interestingly, our preliminary results indicate that RAD54B is phosphorylated in M phase by cyclin-dependent kinases. The candidate will investigate the functional meaning of this post-translational modification.
He/she should also consider other possible post-translational modifications like ubiquitylation and sumoylation.
Milestones: co-immunoprecipitations: 3-6 months; gel filtration: 3 months. Study of post-translational modifications: 3-6 months.
TASK 2: Identification of RAD54B interactors in the presence of DSB lesions.
In order to identify RAD54B interactors, we already started a biochemical approach. We cloned RAD54B in frame with the GST sequence. The peptides are very well expressed and we are setting up the conditions to prepare affinity columns with which the candidate will pull-down putative protein interactors that will then be identified by tandem mass spectrometry (MS/MS), in collaboration with Prof. Corrado Santocanale (NUI Galway). Depending upon the results the candidate will then focus his/her efforts on understanding the functional meaning of the interaction between RAD54B and few interesting putative binding proteins.
Milestones: Biochemical screening: 12 months. Characterization of identified factors: 24 months.
TASK 3: Investigation of the interplay between HELQ, RAD54 and RAD54B at a unique double-strand break in vivo.
In order to study HELQ, RAD54 and RAD54B involvement in HR, the candidate will monitor a single DNA repair event by immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. U2OS-DRGFP cell line that bears a modified GFP gene in which an I-SceI restriction site has been engineered will be used (from Maria Jasin). The candidate PhD student will monitor whether RAD54, RAD54B and HELQ form a single focus, co-localizing with RAD51 and γ-H2AX.
RAD54B, RAD54 and HELQ could bind very close to the break or, being involved in the displacement of RAD51 from dsDNA, they could bind far from the break site. One attractive strategy is to examine the assembly of RAD54B, RAD54 and HELQ on a DSB using ChIP with primer pairs specific to regions of interest proximal or progressively far away from the DSB created by I-SceI.
Milestones: localization at a single DSB: 3-6 months; ChIP analysis: 12 months.