Download Hereditary disorders associated with defects in

Meccanismi di riparazione
Donata Orioli
Risposta cellulare al danno
Ivana Scovassi
RNA
Giuseppe Biamonti
Difetti nei meccanismi di riparazione del
DNA e malattie associate
Donata Orioli/Giovanni Maga
29 Febbraio 2016
Riunione CNAO-CNR-INFN
DNA damaging agents
DNA Repair
systems
endogenous
agents
exogenus
agents
Cellular metabolism
Ionizing radiation
Hereditary disorders associated with defects in
nucleotide-excision repair (NER)
Donata Orioli
Elena Botta
Before
Tiziana Nardo
Fiorenzo A. Peverali
After
Repair by the NER
pathway
Incoming
UV Photon
NER sub-pathways in human cells
Global genome repair
Transcription-coupled repair
Damage recognition
XPC-hHR23B-centrin2
DDB complex
(p127-XPE)
CSA
CSB
TFIIH
Open complex formation
XPG
XPA
RPA
Incision / Excision
DNA repair synthesis
Ligation
ERCC1-XPF
XPG
Clinical and genetic heterogeneity of hereditary
disorders defective in nucleotide-excision repair
XERODERMA PIGMENTOSUM- XP
Cutaneous alterations and tumors in
the sun-exposed areas of the skin
XPA, XPC, XPE, XPF
XP/ CS
XPG
CSA, CSB
COCKAYNE SYNDROME- CS
XPB, XPD
TTDA
TRICHOTHIODYSTROPHY- TTD
Physical and mental retardation
Hair abnormalities
Aging and neurodegeneration
Collection of patient material
•In vitro establishment of cell strains and lines from patients and their relatives;
•Evaluation of the DNA repair defect;
•Identification of the gene/mutations responsible for the pathological phenotype.
• to confirm clinical diagnosis;
• to collect new material from homozygous and heterozygous
carriers with NER defects;
• to identify new NER genes;
• to establish the relationship between clinical symptoms and
molecular defects
Generation of isogenic cell lines to dissect the multiple functions of CSA
and its involvement in Cockayne syndrome
RMCE
CS cells
ROS
UV
irradiation
KBrO3
Menadione
Tandem Affinity Purification
(TAP)
Mass Spectrometry
wtCSAFlag-HA
Flag
HA
E52V-CSAFlag-HA
Flag
HA
Q106P-CSAFlag-HA
Flag
HA
K174A-CSAFlag-HA
Flag
HA
Carla Tribioli, Ennio Prosperi,
Roberta Ricotti, Grace Azar at IGM
Pavia
Heinz Jacobs at The Netherlands Cancer
Institute, Plesmanlaan, Amsterdam.
MOUSE MODELS FOR STUDYING PCNA (Proliferating Cell Nuclear Antigen)
GENE MUTATIONS IN VIVO
SPECIFIC AIM:
1. Production and analysis of Pcna-targeted mouse mutants.
2. To define in vivo the role of Pcna in controlling cell proliferation and tumor
promotion and progression.
3. Possible application as in vivo model to analyze the DNA damage response
pathways induced by hadrontherapic treatments.
Cellular metabolism
Ionizing radiation
Carla Tribioli, Ennio Prosperi,
Roberta Ricotti, Grace Azar at IGM
Pavia
Heinz Jacobs at The Netherlands Cancer
Institute, Plesmanlaan, Amsterdam.
MOUSE MODELS FOR STUDYING PCNA (Proliferating Cell Nuclear Antigen)
GENE MUTATIONS IN VIVO
SPECIFIC AIM:
1. Production and analysis of Pcna-targeted mouse mutants.
2. To define in vivo the role of Pcna in controlling cell proliferation and tumor
promotion and progression.
3. Possible application as in vivo model to analyze the DNA damage response
pathways induced by hadrontherapic treatments.
Ennio Prosperi
IGM-CNR
Research field
DNA damage response:
regulation of DNA repair mechanisms
background
Altered DNA damage response is typical of tumor cells
Malfunction of DNA repair occurs frequently in different human diseases
Aims:
1- to study molecular mechanisms regulating efficiency of DNA repair mechanisms by evaluating the role of :
- protein-protein interactions (interactions between cell cycle regulators and DNA repair factors)
- post-translational modifications (e.g. acetylation of DNA repair factors, such as PCNA, XPG etc.)
2- to evaluate DNA repair efficiency in human syndromes (e.g. Rubinstein-Taybi syndrome with mutations in
CREBBP/EP300 acetyl transferase genes and reduced protein acetylation).
3- to identify action mechanisms of chemical inhibitors of cell proliferation
People:
Francesca Aredia, post-doc
Ilaria Dutto, post-doc
Supported by:
Hereditary disorders associated with defects in
Translesion synthesis (TLS)
Translesion synthesis
DNA Replication
DNA
Replication
14.6% of the XP cases in the Italian population are mutated in the POLH gene and are
affected by the XP variant (XP-V) form
ANALYSIS OF DNA REPLICATION IN PRESENCE OF DAMAGE AND
DAMAGE TOLERANCE
Q: HOW CAN THE CELLS COMPLETE DNA REPLICATION WHEN COMPLEX
DAMAGE IS PRESENT ?
Simone Sabbioneda
We focus our research on DNA polymerases that can specifically bypass a
wide range of DNA adducts
Protein Dynamics (FRAP)
Analysis of nascent DNA
1
14.00
0.8
12.00
10.00
8.00
0.6
XP30RO
wt
6.00
Diffuse polh
Focal polh S phase
Focal polh +HU
Focal polh +UV
0.4
SIM 3
4.00
2.00
0.00
0.2
0
0
0.5 1
1.5 2
2.5 3
Time (s)
Analysis of post-translational modifications
DNA Fibres
DNA Enzymology & Molecular Virology Unit
Hadron therapy - Biological effects
• Double Strand Breaks
• Cluster Lesions
Cellular Response
• Oxidative Lesions
Giovanni Maga
Enzymology of Repair
• DSBs Repair
• Lesion Bypass
Repair DNA Polymerases
In vitro
Emmanuele Crespan
DSBsR – Lesion bypass
In vivo
• Tolerance toward Radiation Therapy
• New Targets – Complementary Chemotherapy/Radiotherapy
Synthetic Lethality
Federico Focher
RNA Epigenetic
Modifications
Survival pathways
DNA repair imbalance
Genomic instability
Malignancy progression
End Joining
pathways
Lesion bypass
Repair DNA pols
Oxidative
damage
tolerance
Hadron Therapy
Cluster Lesions
End Joining
pathways
Lesion bypass
DSBs
Repair DNA pols
Oxidative
Damage
Oxidative
damage
tolerance
Cell Death