DNA repair, recombination, and

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 (MGMT)

other methyltransferases can remove certain methyl groups from methylated adenines and cytosines

Photolyase (only in bacteria and plants) (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 • 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 ) " 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 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 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 • • 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?