DNA repair, recombination, and genome instability
Devon Fitzgerald [email protected] September 7, 2018 TODAY
• DNA damage and repair • Types of DNA damage • DNA repair pathways
• Genome instability • Types of genome alterations • Prevention mechanisms • Cancer development and treatment
• Bioinformatics • Explore cancer ‘omics data in cBioPortal • cBioPortal exercises DNA damage and repair Sources and types of DNA damage
Sources: Alkylating agents Ultraviolet light Ionizing radiation Endogenous processes Carcinogens Radiomimetics
Types:
Single-base damage Base modifications Double-strand (e.g., O6meG) breaks (DSBs) Bulky adducts & Mismatches intra-strand crosslinks DNA damage is very common Overview of DNA repair
Single-base Base modifications Double-strand damage (e.g., O6meG) breaks (DSBs) Bulky adducts & Mismatches intra-strand crosslinks Homology- directed repair Base excision Direct reversal (DR) (HDR) repair (BER) Non-homologous Nucleotide excision Mismatch end joining repair (NER) repair (MMR) (NHEJ) Direct reversal (DR)
Base modifications (e.g., O6meG)
Direct reversal (DR) Direct reversal (DR)
Methyl guanine methyl transferase (MGMT)
other methyltransferases can remove certain methyl groups from methylated adenines and cytosines
Photolyase (only in bacteria and plants) Base excision repair (BER)
Single-base damage
Base excision repair (BER) Base excision repair (BER)
Hegde et. al. (2012) Biomolecules Base excision repair (BER): E. coli
DNA glycosylases • Ung, Mug, AlkA, MutY, Tag • Fpg, Nei, Nth
AP endonucleases • Xth • Nfo
DNA polymerase • Pol I
Flap endonuclease • Pol I
DNA ligase • DNA Ligase (LigA) Base excision repair (BER): human
DNA glycosylases • UNG (uracil) • OGG1 (8-oxoG, FapyG) • NEIL1 (Tg, hoU, hoC, urea, FapyG, FapyA)
AP endonucleases • APE1 • APE2
DNA polymerases • Short-patch: Pol ! • Long-patch: Pol " and Pol # (with PCNA)
Flap endonuclease • FEN1 (long-patch only)
DNA ligase • LIG3 (with XRCC1) Nucleotide excision repair (NER)
Bulky adducts & intra-strand crosslinks
Nucleotide excision repair (NER) Nucleotide excision repair (NER)
Lesion recognition (E. coli) • TC-NER: RNAP, Mfd & UvrAB • GG-NER: UvrAB
Unwinding (E. coli) • UvrB (with UvrA)
Cleavage (E. coli) • UvrC (with UvrB) • UvrD removes fragment
DNA synthesis (E. coli) • DNA Pol I (PolA)
Ligase (E. coli) • DNA ligase (LigA) Nucleotide excision repair (NER)
Lesion recognition (human) • TC-NER: RNA polymerase II • GG-NER: XPC & RAD23B
Unwinding (human) • TFIIH (with XPA, XPG & RPA)
Cleavage (human) • XPG • XPF/ERCC1
DNA synthesis (human) • Pol ! (with PCNA) • Pol " (with PCNA)
Ligase (human) • LIG1 Nucleotide excision repair (NER)
UV lesions recognized 2 ways
• Global genomic NER (GG-NER)
• Transcription-coupled NER (TC-NER) Damage tolerance: TLS polymerases
Translesion synthesis (TLS) polymerases
• can replicate damaged DNA normally repaired by DR, BER, or NER
• introduce many mutations due to mis- pairing with damaged bases and lack of proof-reading activity
• “emergency back-up” if DNA must be replicated before repair has occurred
• programmed use in antibody maturation (somatic hypermutation) Mismatch repair (MMR)
Mismatches
Mismatch repair (MMR) Mismatch repair (MMR) Mismatch repair (MMR)
E. coli MutS = human MSH2, MSH3, MSH6
E. coli MutL = human MLH1, MLH3, PMS1, PMS2
E. coli MutH = human ??
E. coli UvrD = human ??
E. coli SSB = human SSB
E. coli Exo I (?) = human Exo I
E. coli Pol III ≈ human Pol !
Nimesh et al (2006) DNA double-strand break repair
Double-strand breaks (DSBs)
Homology- directed repair (HDR)
Non-homologous end joining (NHEJ) DNA double-strand breaks (DSBs)
Goodarzi & Jeggo (2013) Advances in Genetics DSB repair: two major pathways
! Quick and efficient
! No homolog required ! Higher fidelity . (can occur throughout cell cycle) " Requires homologous template " Lower fidelity . (can only occur in S or G2) Non-homologous end joining (NHEJ)
End recognition (human) • Ku70/Ku80 • DNA-PKcs
End processing (human) • Pol ! & Pol "
Ligase (human) • Ligase IV (with XRCC4) NHEJ in class-switch recombination
Class-switch recombination changes the genome of B cells so that different antibody isotypes can be produced
AID make programmed DSBs in activated B cells
DSBs are repaired by NHEJ
Jolly et al (2008) J. of Experimental Medicine Homology-directed repair (HDR) End resection E. coli: RecBCD human: MRN (Mre11, Rad50, Nbs1)
Recombinase loading E. coli: RecBCD human: BRCA2 Strand invasion RecA/Rad51 Repair synthesis (recombinase) Holiday junction resolution E. coli: RuvABC human: GEN1 or MUS81-EME1 Homology-directed repair (HDR)
Synthesis-dependent strand annealing (SDSA) Homology-directed repair (HDR)
Break-induced replication
• Processive (continues for very long distances) • Conservative DNA replication, de-coupled leading and lagging strand synthesis • Very error-prone (makes many mutations) • Causes loss-of-heterozygosity HDR of programed DSBs in meiosis
Recombination promotes genetic diversity during sexual reproduction • Cross-overs and gene conversions allow gametes to contain a mix of parental alleles HDR of programed DSBs in meiosis
Programmed DSBs made by Spo11
Mirzaghaderi & Hörandl (2016) Royal Proc. B HDR of other DNA damage Summary: DNA damage and repair Summary: DNA damage and repair
Weeden & Asselin-Labat (2018) BBA Molec. Basis of Disease Nobel Prize in Chemistry 2015 Genome Instability Types of genome variations
http://www.ensembl.org/info/genome/variation/index.html Point mutations
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%207/mutation.html Chromosomal rearrangements
Alqallaf et al (2013) Recent Advances in Autism Spectrum Disorders Preventing genome instability
Shen et al (2011) Journal of Molecular Cell Biology Preventing genome instability
CANCER Diseases caused by DNA repair defects
(HNPCC) Genome instability in cancer
Negrini et al (2010) Nature Reviews Genome instability in cancer
Most cancers show high mutation burden -> genome instability
Is genome instability a cause or a consequence of cancer? BOTH! Mutations drive cancer evolution
Uchi et al (2016) PLoS Genetics MMR proteins as tumor suppressors
MMR prevents mutations by repairing errors made during DNA replication
MMR also promotes genome stability through other pathways
Germline loss-of-function mutations in MMR genes cause Hereditary Non-Polyposis Colon Cancer (HNPCC)
Somatic MMR gene mutations are common in many types of sporadic cancer
Li & Martin (2016) Trends in Molecular Medicine BRCA1 & BRCA2 as tumor suppressors
BRCA1 & BRCA2 promote high-fidelity HDR of DSBs and proper cell cycle checkpoints
Loss of BRCA1 or BRCA2 disrupts these processes, causing genome instability
BRCA1/2+/- -> Very high cancer disposition (especially breast and ovarian) Exploiting genome instability Exploiting genome instability: PARPi
Polyak & Garber (2011) Nature Exploiting genome instability: more But...mutations drive evolution
increased genetic diversity = Uchi et al (2016) PLoS Genetics increased risk of therapy resistance More damage = more heterogeneity
Fedele et al (2014) Cancer Discovery More damage = future cancer risk
Etoposide causes DSBs and is associated with subsequent development of AML
Etoposide-induced DSBs are at common AML-driving rearrangement breakpoints
Canela et al (2017) Cell Alternative strategy: anti-evolvability drugs Bioinformatics Cancer ‘omics data in cBioPortal
• Web-based tool to explore the increasing number of cancer ‘omics datasets • Very large studies with many data types: • Genome/exome sequencing • Copy-number analysis • Gene expression • Proteomics • Metabolomic Cancer ‘omics data in cBioPortal
http://www.cbioportal.org/ Cancer ‘omics data in cBioPortal BRCA1 & 2 mutations in ovarian cancer BRCA1 & 2 mutations in ovarian cancer BRCA1 & 2 mutations in ovarian cancer BRCA1 & 2 mutations in ovarian cancer
This view is more interesting for pan-cancer analysis! BRCA1 & 2 mutations in ovarian cancer BRCA1 & 2 mutations in ovarian cancer BRCA1 & 2 mutations in ovarian cancer BRCA1 & 2 mutations in ovarian cancer Querying cBioPortal: select studies Select genomic profiles, case set, gene(s)
pick a gene set OR enter gene(s) Exercise: TP53 in pan-cancer analysis
• What percentage of the total cases have some kind of alteration in TP53?
• What cancer type has the highest frequency of TP53 alterations?
• Did patients with a TP53 mutation have a better or worse clinical outcome (survival)? Exercise: TP53 in pan-cancer analysis
• What percentage of the total cases have some kind of alteration in TP53? 43% • What cancer type has the highest frequency of TP53 alterations? Small cell lung cancer • Did patients with a TP53 mutation have a better or worse clinical outcome (survival)? worse EXPLORE! Choose your own adventure Exercise: DNA repair pan-cancer analysis
• Use the following pan-cancer study: • MSK-IMPACT Clinical Sequencing Cohort (MSKCC, Nat Med 2017)
• Pick one of the following gene sets: • NEIL1 & FEN1 (BER) • XPC & XPG (NER) • MSH2 & MLH1 (MMR) • BRCA1 & BRCA2 (HDR) Exercise: DNA repair pan-cancer analysis
• Which gene is altered more frequently?
• Which cancer type has the highest frequency of alterations?
• Do either of your genes of interest have mutation hot-spots?
• How do alterations in your genes of interest affect survival?