Of complexes and maintenance of genome stability Marek Sebesta, PhD marek.sebesta@ceitec.muni.cz CSB, Ceitec, MU 24-Mar-22 Content 1. What is maintenance of genome stability? 2. What are the challenges to the genome stability? 3. How do cells know the genome stability has been compromised? 4. How do cells maintain the genome stability? 5. How to study the genome stability maintenance? (Case study on Homologous recombination) 2 What is the maintenance of genome stability? 3 3 What is the maintenance of genome stability? What is the maintenance of genome stability? 3 What is the maintenance of genome stability? 3 What is the maintenance of genome stability? 3 What is the maintenance of genome stability? 4 What is the maintenance of genome stability? It is the ability of living organisms to preserve its genetic material in time and across generations. 4 What are the challenges to genome stability? All living mater is constantly exposed to environment that challenges genome stability Ribosome Centriole 5 What are the challenges to genome stability? All living mater is constantly exposed to environment that challenges genome stability Endogenous Cellular metabolism DNA replication Transcription Spontaneous modification of the DNA Ribosome Mitochondrion Cytoplasm Lysosome Smooth endoplasmic reticulum Rough endoplasmic reticulum Plasma membrane Cell coat Free ribosome Centriole Nucleus Nucleolus Chromatin Nuclear pore Nuclear envelope Colgi body 5 What are the challenges to genome stability? All living mater is constantly exposed to environment that challenges genome stability Endogenous Cellular metabolism DNA replication Transcription Spontaneous modification of the DNA Ribosome Mitochondrion Cytoplasm Lysosome Smooth endoplasmic reticulum Rough endoplasmic reticulum Plasma membrane Cell coat Free ribosome Centriole Nucleus Nucleolus Chromatin Nuclear pore Nuclear envelope Colgi body Exogenous Radiation Diet Stress 5 Damaging agent X-rays Oxygen radicals Alkylating agents Spontaneous reactions UV light Polycyclic aromatic hydrocarbons X-rays Anti-tumour agents (c/'s-Pt, MMC) Replication errors Uracil Abasie site 8-Oxoguanine Single-strand break (6-4)PP Bulky adduct CPD Interstrand cross-link Double-strand break A-G Mismatch T-C Mismatch Insertion Deletion + Base-excision repair (BER) Nucleotide-excision repair (NER) Recombinational repair (HR, EJ) Mismatch repair Repair process Hoeijmakers, 2001 6 What are the challenges to genome stability? Damaging agent ♦ X-rays Oxygen radicals Alkylating agents Spontaneous reactions UV light Polycyclic aromatic hydrocarbons X-rays Anti-tumour agents (c/'s-Pt, MMC) Uracil Abasie site 8-Oxoguanine Single-strand break (6-4) PP Bulky adduct CPD Interstrand cross-link Double-strand break Base-excision repair (BER) Nucleotide-excision repair (NER) Recombinational repair (HR, EJ) Repair process Replication errors A-G Mismatch T-C Mismatch Insertion Deletion + Mismatch repair Hoeijmakers, 2001 6 What are the challenges to genome stability? 7 What are the challenges to genome stability? What is more prevalent? Exogenous or endogenous damage? What are the challenges to genome stability? What is more prevalent? Exogenous or endogenous damage? Even-though, historically, exogenous DNA damage was considered to be the prime cause of mutagenesis, recently, as the methodology has progressed, the cellular DNA metabolism pathways (replication and transcription) are being recognised as the more prevalent cause of mutations. 7 What are the challenges to genome stability? Inability to repair properly the damage may lead to cancer, senescence, apoptosis. exogenous endogenous Mmage °0° Metabolism nuclear DNA \ unrepaired cancer mitochondrial DNA unrepaired senese^ce apoptosis healthy cell rare of DNA damage = rate or repair up to 500.000 DNA modiftcation events per cell per day diseased cell rate or DNA damage > rate or repair 8 What is the difference between a primary lesion and a mutation? 9 What is the difference between a primary lesion and a mutation? (Carr 1999) .G. 4 ■ p. 2?- | | Oxidative Damage 9 What is the difference between a primary lesion and a mutation? (Carr 1999) .G. 4 Oxidative Damage I ■G. Excision Repair Standard Allele 9 What is the difference between a primary lesion and a mutation? (Carr 1999) Replication .G. i ' C ' .GO. TTCT1 J_J_G ^ next replication rr J_LGOJ_L .G. i T Oxidative Damage I -G. Excision Repair Standard Allele transversion Standard Allele 9 What is the difference between a primary lesion and a mutation? (Carr 1999) "C .G primary lesion TTCTl J_LG ^ next replication I Excision Repair Standard Allele transversion Standard Allele 10 What is the difference between a primary lesion and a mutation? mutation 10 Transient summary I 11 Transient summary I Terms Genome stability, DNA damage response, DNA repair, DNA damage tolerance denote closely related, yet not interchangeable terms 11 Transient summary I Terms Genome stability, DNA damage response, DNA repair, DNA damage tolerance denote closely related, yet not interchangeable terms Cells are continuously exposed to wide variety of DNA damage 11 Transient summary I Terms Genome stability, DNA damage response, DNA repair, DNA damage tolerance denote closely related, yet not interchangeable terms Cells are continuously exposed to wide variety of DNA damage Failure to properly deal with the damage may have fatal consequences to cells 11 How do cells know genome stability has been compromised? 12 How do cells know genome stability has been compromised? X-rays Oxygen radicals Alkylating agents Spontaneous reactions Damaging agent UV light Polycyclic aromatic hydrocarbons X-rays Anti-tumour agents (c/s-Pt, MMC) Replication errors Uracil Abasie site 8-Oxoguanine Single-strand break (6-4) PP Bulky adduct CPD Interstrand cross-link Double-strand break A-G Mismatch T-C Mismatch Insertion Deletion t Base-excision repair (BER) Nucleotide-excision repair (NER) Recombinational repair (HR, EJ) Mismatch repair Repair process Hoeijmakers, 2001 12 How do cells know genome stability has been compromised? The challenges - different types of DNA damage 13 How do cells know genome stability has been compromised? The challenges - different types of DNA damage - cell-cycle stage 13 Hoeijmakers, 2001 How do cells know genome stability has been compromised? The challenges - different types of DNA damage - cell-cycle stage Hoeijmakers, 2001 How do cells know genome stability has been compromised? The challenges different types of DNA damage cell-cycle stage metabolic state Hoeijmakers, 2001 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA Down-stream events 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA Down-stream events 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA A B RNAPII CSB Down-stream events Down-stream events 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA Down-stream events J Down-stream events 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA A B RNAPII CSB Down-stream events Down-stream events C DNA Pol 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA A CSB Down-stream events Down-stream events C DNA Pol case 14 How do cells know genome stability has been compromised? Cells possess context-specific sensors that recognise signals from the damaged DNA A CSB Down-stream events Down-stream events C DNA Pol case Down-stream eventSi 4 How do cells react to DNA damage? DNA damage Replication stress Signal Sensors Transducers Damage repair m- Effectors Transcription Cell cycle transitions Apoptosis 15 How do cells react to DNA damage? A simplified picture Ionizing radiation DNA damage sensors Apical kinases DNA damage mediators Downstream kinases Effectors Outcomes Oncogene activation DNA replication stress (ET MRE11-RAD50-NBSO ( RPA ) ? \\0 J a ATR t -qx P RAD9-RAD1-HUS1 ami CHK2 T CHK1 P53 Apoptosis Checkpoint arrest \ DNA repair No DNA repair Proliferation Cellular senescence 16 d'Adda d'Fagagna, 2012 How do cells react to DNA damage? A more comprehensive picture 17 Aguilera and Garcia-Muse, 2013 Transient summary II 18 Transient summary II Cells possess specific factors - sensors - that recognise insults to DNA structure, DNA breaks, or stalled machineries like transcription and replication. Transient summary II Cells possess specific factors - sensors - that recognise insults to DNA structure, DNA breaks, or stalled machineries like transcription and replication. The sensors subsequently activate complex signalling pathways that lead to halt of cell-cycle, as well as to decision as of which pathway is to be used; balancing the cell-cycle stage and other needs of the cell. 18 How do cell maintain genome stability? DNA repair is prevalent outside the S-phase, in which DNA damage tolerance is preferred. Hoeijmakers, 2001 How do cell maintain genome stability? Endogenous or environmental SAM Nitrosated amines and bile acids Dietary nitrosamines • ROS • SAM • Natural IR • Base deamination or loss • Trapped TOPOI ROS UV Tobacco smoke Afflatoxin Replication errors SAM Base deamination • ROS • Natural IR • Trapped TOPOII Unrepaired single strand lesions • Acrolein • Croton-aldehyde Lesion 10-30 06-methyl-guanine 10,000-100,000 • 8-oxoguanine • N7-meG • N3-meA • Uracil • Hypoxanthine • Xanthine •SSB • 6-4 photo-products • Cyclopurines • Bulky adducts Base mismatches DNA double-strand breaks 10-50 Stalled replication forks ICL Therapeutic •TMZ • Alkylating agents • Nitrosoureas •TMZ • IR • Radiomimetics • TOPOI poisons • Antimetabolites • Cisplatin • Carboplatin • Nitrosoureas •TMZ • Nucleoside analogues • IR • Radiomimetics • TOPOI poisons TMZ TOPOI poisons Antimetabolites Cisplatin Carboplatin Nitrosoureas MMC Repair pathway BER SSBR NHEJ V Curtin et al., 2012 20 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Sebesta and Krejci, 2016 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Sebesta and Krejci, 2016 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB -> i NHEJ NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Sebesta and Krejci, 2016 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair D-loop TV gene conversion SDSA Sebesta and Krejci, 2016 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Sebesta and Krejci, 2016 D-loop 7\ 1 dHJ dissolution gene conversion SDSA gene conversion crossover DSBR gene 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Sebesta and Krejci, 2016 D-loop 7\ 1 dHJ dissolution gene conversion SDSA gene conversion crossover DSBR gene 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ Error-prone NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Sebesta and Krejci, 2016 D-loop 7\ 1 dHJ dissolution gene conversion SDSA gene conversion crossover DSBR gene 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair DSB NHEJ Error-prone NHEJ: non-homologous end joining SSA: single strand annealing SDSA: synthesis-dependent strand-annealing DSBR: DSB repair Error-free Sebesta and Krejci, 2016 D-loop 7\ 1 dHJ dissolution gene conversion SDSA gene conversion crossover DSBR gene 21 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Non-homologous end joining 22 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Non-homologous end joining 22 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Non-homologous end joining 22 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Non-homologous end joining NHEJ is an error-prone pathway Restoration of DNA integrity 22 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Pre - synapsis Sebesta and Krejci, 2016 23 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination ^^^^^^^ Pre-synapsis O Rad51(RAD51) Nucleosome MRX, MRN bridging, trimming c@g) MRX(MRN) T and opening of ends Sebesta and Krejci, 2016 23 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Q> RPA O Rad51(RAD51) Nucleosome MRX(MRN) Pre-synapsis MRX, MRN bridging, trimming and opening of ends Sgs 1 -Top3-Rmi 1 /Dna2/RPA BLM-TOPIIIa-RMI1-RMI2/DNA2/RPA Exo1 EX01 long range resection ^ÖÖQQ^ RPA loading Sebesta and Krejci, 2016 23 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Q> RPA O Rad51(RAD51) Nucleosome MRX(MRN) Pre-synapsis MRX, MRN bridging, trimming and opening of ends Sgs 1 -Top3-Rmi 1 /Dna2/RPA BLM-TOPIIIa-RMI1-RMI2/DNA2/RPA Exo1 EX01 long range resection ^ÖÖQQ^ RPA loading Rad51 nucleation and stabilization Rad52/Rad59 (Rad55-57, Shu complex) BRCA2/RAD52 (RAD51 paralogies) It Rad51 removal Srs2 RECQ5, FBH1, PARI Sebesta and Krejci, 2016 Rad51 nucleoprotein filamenr-^-structure able to perform homology search How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Synapsis Homology search O Rad51 (RAD51) £B Histones Rad54 (RAD54) Ino80 Polymerase Sebesta and Krejci, 2016 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Synapsis Homology search A RTEL Mph1, FANCM -D-loop dissasembly Rad54, RAD54-dependent homology search Ino80, increased chromatin mobility DNA damage checkpoint-mediated phosphorylation D-loop formation O Rad51 (RAD51) Histones Rad54 (RAD54) Q Ino80 Polymerase Sebesta and Krejci, 2016 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Synapsis Homology search RTEL Mph1, FANCM -D-loop dissasembly Rad54, RAD54-dependent homology search \^ Ino80, increased chromatin mobility DNA damage checkpoint-mediated phosphorylation D-loop formation POLk, (POLr|) potentially PCNA-independent O Rad51 (RAD51) Histones Rad54 (RAD54) Pol5 (Polr|) POL5 (POLr|) PCNA-dependent Ino80 Polymerase Sebesta and Krejci, 2016 Shorter extension Longer extension How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Post- synapsis D-loop Sebesta and Krejci, 2016 25 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Post- synapsis Mph1 (Srs2?, Irc20?) FANCM, RTEL D-loop gene conversion SDSA Sebesta and Krejci, 2016 25 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Post- synapsis Mph1 (Srs2?, Irc20?) FANCM, RTEL D-loop 1 Rad52 I' dHJ _ I I Mus81/Mms4, Slx1/Slx4, Yen1, Mlh1, Exo1 MUS81/EME1, SLX1/SLX4, GEN1, MLH1, EX01 gene conversion crossover DSBR Sebesta and Krejci, 2016 25 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Post- synapsis D-loop ^Rad52 Mph1 (Srs2?, Irc20?) FANCM, RTEL Sgs1-Top3-Rmi1 BLM-TOPIIIa-RMI1-RMI2 Mus81/Mms4, Slx1/Slx4, Yen1, Mlh1, Exo1 MUS81/EME1, SLX1/SLX4, GEN1, MLH1, EX01 gene conversion crossover DSBR Sebesta and Krejci, 2016 25 How do cell maintain genome stability? Double-stranded DNA breaks (DSB) repair Homologous recombination Post- synapsis Single-ended DSB (e.g. collapsed fork) Mph1 (Srs2?, Irc20?) FANCM, RTEL Partial RF half crossover BIR Sebesta and Krejci, 2016 Sgs1-Top3-Rmi1 BLM-TOPIIIa-RMI1-RMI2 Mus81/Mms4, Slx1/Slx4, Yen1, Mihi, Exo1 MUS81/EME1, SLX1/SLX4, GEN1, MLH1, EX01 gene conversion crossover DSBR 25 Transient summary III 26 Transient summary III Different types of DNA damage are repaired by specific repair pathway 26 Transient summary Different types of DNA damage are repaired by specific repair pathway The repair is generally error-free, except for NHEJ and SSA 26 Transient summary III Different types of DNA damage are repaired by specific repair pathway The repair is generally error-free, except for NHEJ and SSA In S-phase, cells activate tolerance mechanisms that allow timely completion of DNA replication 26 How to study genome stability maintenance? (Case study on Homologous recombination) Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes Hannah L Klein1*, Kenny K.H. Ang2, Michelle R. Arkin2, Emily C. Beckwitt3'4, Yi-Hsuan Chang5, Jun Fan6, Youngho Kwon7'8, Michael J. Morten1, Sucheta Mukherjee9, Oliver J. Pambos6, Hafez el Sayyed6, Elizabeth S. Thrall10, Joäo P. Vieira-da-Rocha9, Quan Wang11, Shuang Wang1213, Hsin-Yi Yeh5, Julie S. Biteen14, Peter Chi515, Wolf-Dietrich Heyer9'16, Achillefs N. Kapanidis6, Joseph J. Loparo10, Terence R. Strick121317, Patrick Sung7'8, Bennett Van Houten31819, Hengyao Niu11* and Eli Rothenberg1* Review eel www.microbialcell.com 27 How to study genome stability maintenance? (Case study on Homologous recombination) Different strategies exist Genetic tools Enable us to identify genes and the relationships among, thereby building a pathway Microscopic tools Give us a glimpse at spacial and temporal relationships of genes of interests Biochemical tools Structural tools Enable us to understand mechanisms and complex formations within a studied pathway Enable us to understand molecular mechanisms at atomic resolution Single molecule techniques Enable us to understand behaviour at of single molecules as compared to bulk biochemical reactions 28 How to study genome stability maintenance? Stepl: identify the genes Molec. gen. Genet. 125, 197—216 (1973) © by Springer-Verlag 1973 Interactions among Genes Controlling Sensitivity to Radiation and Alkylation in Yeast Martin Brendel and Robert H. Haynes Department of Biology, York University, Toronto, Canada Received March 27, 1973 29 How to study genome stability maintenance? Stepl: identify the genes Molec. gen. Genet. 125, 197—216 (1973) © by Springer-Verlag 1973 Interactions among Genes Controlling Sensitivity to Radiation and Alkylation in Yeast Martin Brendel and Robert H. Haynes Department of Biology, York University, Toronto, Canada Received March 27, 1973 Using a thorough genetic analysis of the isolated mutants, they were able to build a first model of multiple pathways dealing with DNA damage. 1 i /uv-DNA ) [ HN2-DNA/ 'X-ray-DNA MMS-DNA uvsKRad Quvs 9 (Rad 2) Excision repair (Rad 3), (Rad 4) uxsl (Rad 18) Post replication repair ? xsi 'X- Repair' p 2 Not characterized at molecular level 29 How to study genome stability maintenance? Stepl: identify the genes nature vd 455l9 October 2008|doi:10.1038/nature07312 ARTICLES Sae2r Exol and Sgsl collaborate in DNA double-strand break processing Eleni P. Mimitou' & Lorraine S. Symington1 30 How to study genome stability maintenance? Stepl: identify the genes nature v # $ r7-r6v r4; r3x ---- r2 - —- ----- --- r1- ----- Uncut Cut ^_____ ___^ — * ---- *** _ 3' specific single-stranded probe Uncu! Cu - ] 5' specific single-stranded probe Figure 3 | Single-stranded intermediates fail to form in the absence of Exol and Sgsl. a, Representation of the method used to detect single-stranded r5-r4-r3-r2-r1 - HO 30 How to study genome stability maintenance? Stepl: identify the genes nature Vol 45519 October 2008|doi:10.1038/nature07312 ARTICLES Sae2, Exol and Sgsl collaborate in DNA double-strand break processing Eleni P. Mimitou1 & Lorraine S. Symington1 Recruitment of MRX/Sae2 to the DSB I I Trimming of ends by MRX/Sae2 -3' 3-- -3' ► Exo1 3'- Processive resection by Exo1 and/or Sgs1 Figure 5 | Two-step mechanism for DSB resection. After a DSB is formed Using a genetic approach Mimitou and Symington, were able to show for the first time the mechanism by which cells resect the ends of broken DNA. 30 r7. r6-r5-r4-r3. r2-r1 - _i 10 kb -= 6.8 kb -»6.1 kb -"5.7 kb -a 5 kb -"2.7 kb HO —=1.6kb : Cut 0.7 kb : Uncut 0.9 kb i ss probe W X Ya Z1 Z2h S B B A A A A A S S B S/B S rad51A b rad51A rad51A exol A rad51A sgsIA exolA sgslA Time (min): o <$$jP xl-r6v r4; r3x r2 M- Uncut Cut - —- --- ___m—* ---- _ 3' specific single-stranded probe - ] 5' specific single-stranded probe Figure 3 | Single-stranded intermediates fail to form in the absence of Exol and Sgsl. a, Representation of the method used to detect single-stranded How to study genome stability maintenance? Step2: purify and study the proteins alone Catalysis of ATP-Dependent Homologous DNA Pairing and Strand Exchange by Yeast RAD51 Protein Patrick Sung «- RAD51 *- rad51* Fig. 1. Overproduction and purification of RAD51 protein. (A) Immunoblot analysis. The nitrocellulose blot of a 9% denaturing polyacrylamide gel was probed with affinity-purified antibodies to RAD51. Lane 1, extract from the rad51 deletion yeast strain YR51A-1; lane 2, extract from the yeast strain LP2749-9B harboring the 2\l multicopy vector pSCW231, which contains the ADC 7 promoter but lacks the RAD51 gene; lane 3, extract from strain LP2749-9B harboring the 2p. plasmid pR51.2, which contains the RAD51 gene under the control of its own promoter; lane 4, extract from strain LP2749-9B harboring the 2jt plasmid pR51.1, which contains the RAD51 gene under the control of the ADC1 promoter; and lane 5,10 ng of purified RAD51 protein. (B) Purity analysis by SDS-PAGE. A 9% denaturing polyacrylamide gel was stained with Coomassie blue. Lane 1, molecular size markers; lanes 2 and 3,1 fig and 3 p.g of purified RAD51 protein. Molecular sizes are indicated on the left (in kilodaltons). A KB 97 — 66- 43- 31- B kJ2 97 — 66 — 43 — 31 — 21 — 31 How to study genome stability maintenance? Step2: purify and study the proteins alone Catalysis of ATP-Dependent Homologous DNA Pairing and Strand Exchange by Yeast RAD51 Protein Patrick Sung A - RAD51 -rad51* - RAD51 «- rad51* Fig. 1. Overproduction and purification of RAD51 protein. (A) Immunoblot analysis. The nitrocellulose blot of a 9% denaturing polyacrylamide gel was probed with affinity-purified antibodies to RAD51. Lane 1, extract from the rad51 deletion yeast strain YR51A-1; lane 2, extract from the yeast strain LP2749-9B harboring the 2(x multicopy vector pSCW231, which contains the ADC 1 promoter but lacks the RAD51 gene; lane 3, extract from strain LP2749-9B harboring the 2|i. plasmid pR51.2, which contains the RAD51 gene under the control of its 1 z 3 own promoter; lane 4, extract from strain LP2749-9B harboring the 2(jl plasmid pR51.1, which contains the RAD51 gene under the control of the ADC1 promoter; and lane 5,10 ng of purified RAD51 protein. (B) Purity analysis by SDS-PAGE. A 9% denaturing polyacrylamide gel was stained with Coomassie blue. Lane 1, molecular size markers; lanes 2 and 3,1 ^.g and 3 jj.g of purified RAD51 protein. Molecular sizes are indicated on the left (in kilodaltons). 97— 66 — 43- 31- B kß 97 — 66 — 43 — 31 — 21 — 0 (+) Synapsis Viral ss Linear ds 4 (-) Strand exchange -► RAD51 - + + + Time (min) 60 10 30 60 Joint molecule 60 Nicked circular Displaced ss RAD51 Time (min) 60 Displaced ss- Displaced ss 3' Labeled 5' Labeled Using a purified protein, Patrick Sung was able to show that Rao!51 is a bona fide recombinase. 31 How to study genome stability maintenance? Step2: purify and study the proteins in assemblies nature Vo1 467l2 September 2010|doi:10.1038/natureO9355 LETTERS DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2 Petr Cejka1'2, Elda Cannavo1 '2, Piotr Polaczek3, Taro Masuda-Sasa3, Subhash Pokharel3, Judith L. Campbell3 & Stephen C. Kowalczykowski1'2 32 How to study genome stability maintenance? Step2: purify and study the proteins in assemblies nature Vol 467|2 September 2010|doi:10.1038/nature09355 LETTERS DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2 Petr Cejka1'2, Elda Cannavo1,2, Piotr Polaczek3, Taro Masuda-Sasa3, Subhash Pokharel3, Judith L. Campbell3 & Stephen C. Kowalczykowski1'2 co E c ■. >■ # • • ■ Rdh54-YFP Mtw1-CFP Rad52-RFP DIC • YC merge YR merge CR merge YCR merge •i <• • • -a. U* % • t* * Ddd-YFP Rad52-CFP merge DIC • • • • Rad9-YFP Rad52-CFP merge DIC • ■ • A • Mre11-YFP Rfal-CFP merge DIC .a. 1 r f g Ddc2-YFP Rfa1-CFP merge DIC 33 Figure 1. Colocalization of Checkpoint and Repair Foci How to study genome stability maintenance? Step3: study the proteins in time and space Cell, Vol. 118, 699-713, September 17, 2004, Copyright ©2004 by Cell Press Choreography of the DNA Damage Response: Spatiotemporal Relationships among Checkpoint and Repair Proteins Michael Lisby,1'3 Jacqueline H. Barlow, Rebecca C. Burgess,2 and Rodney Rothstein* E DSB formation time MRX and Tell 3ae2 RP-A, Med/Ddc2. Rad24/Rfc2-5 and Ddc1/Mec3'Rad17 Rad52. Rad59. Rad51, Rdh54. Rad55 and Rad54 DSB ends Mre11- / red single-stranded DNA Rad24 Pfc2-5 Ddc1/ Pi-!-; Using life-cell microscopy, Lisby et al., were able to study the spatiotemporal interactions among recombination factors. Rad59-YFP Rad52-CFP merge DIC • >■. >■ # • • ■ Rdh54-YFP Mtw1-CFP Rad52-RFP DIC • YC merge YR merge CR merge YCR merge •i <• • P « • -a. U* % • t* * Ddd-YFP Rad52-CFP merge DIC • • • • Rad9-YFP Rad52-CFP merge DIC • ■ • A • Mre11-YFP Rfal-CFP merge DIC .a. 1 r f f Ddc2-YFP Rfa1-CFP merge DIC Rad54 33 Figure 1. Colocalization of Checkpoint and Repair Foci How to study genome stability maintenance? Step4: study the role of protein complex formation? Protein Group Modification and Synergy in the SUMO Pathway as Exemplified in DNA Repair Ivan Psakhye1 and Stefan Jentsch1* department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany 'Correspondence: jentsch@biochem.mpg.de http://dx.doi.Org/10.1016/j.cell.2012.10.021 A Using SILAC approaches, Psakhye and Jentsch showed that majority of HR proteins are Sumoylated upon DSBs induction. Light LysOArgO Untreated i- Heavy Lys8Arg10 MMS 0.2% "•■•SUMO Ni-NTA PC k0 »0-ix-100- 75- W- 37- »-20 -I» - 1 3 3 « t-_. • 9 it.- ic "o 8 I Trypbc digestion. LC-MS/MS, MASCO T/MaxQuant data processing RFA1 •jl .» • RAD52 , RAD9 RAD59 ' MRC1 RFA2 -+- 0 5 SILAC rabo (log2 MMS-treated/untreated) 10 34 How to study genome stability maintenance? Step4: study the role of protein complex formation? Protein Group Modification and Synergy in the SUMO Pathway as Exemplified in DNA Repair Ivan Psakhye1 and Stefan Jentsch1* 1Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany "Correspondence: jentsch@biochem.mpg.de http://dx.doi.Org/10.1016/j.cell.2012.10.021 YPD 002% MMS 1 ;. 1 - : 5 - 0 10 Troe n Tuna.h This Sumo-SIM mediated interactions are trigger timely completion of HR. 35 How to study genome stability maintenance? Step4: study the role of protein complex formation? Protein Group Modification and Synergy in the SUMO Pathway as Exemplified in DNA Repair Ivan Psakhye1 and Stefan Jentsch1 * 1 Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany 'Correspondence: jentsch@biochem.mpg.de http://dx.doi.Org/10.1016/j.cell.2012.10.021 This Sumo-SIM mediated interactions are trigger timely completion of HR. Chromatin-associated SUMOylation machinery I SIZ2 Protein-group SUMOylation fosters complex formation and DNA repair 36 How to study genome stability maintenance? Step5: study the molecular mechanisms by the means of structural biology Vol 453|22 May 2008|doi:10.1038/nature06971 ARTICLES Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures Zhucheng Chen1,1, Haijuan Yang1 & Nikola P. Pavletich1" Crystal structure of presynaptic filament ADP-AIF4-Mg ^ 37 How to study genome stability maintenance? Step5: study the molecular mechanisms by the means of structural biology Vol 453|22 May 2008|doi:10.1038/nature06971 ARTICLES Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures Zhucheng Chen1,1, Haijuan Yang1 & Nikola P. Pavletich1,2 Crystal structure of postsynaptic filament. ADP-AIF4-Mg RecA5 38 How to study genome stability maintenance? Step5: study the molecular mechanisms by the means of structural biology Vol 453|22 May 2008|doi:10.1038/nature06971 nature ARTICLES Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures Zhucheng Chen1,1, Haijuan Yang1 & Nikola P. Pavletich1,2 By comparing the two structure a detailed, molecular mechanism of the strand exchange reaction can be inferred. 39 How to study genome stability maintenance? Step6: study the molecular mechanisms by the means of single-molecule techniques. DNA RECOMBINATION Base triplet stepping by the Rad51/RecA family of recombinases Ja YD Lee,1 Tsuyoshi Terakawa,12* Zhi Qj,1* Justin B. Steinfeld,1 Sy Redding,1 YounglIo Kwon,4 William A. Gaines,4 YVcixing Zhao,4 Patrick Sung,4 Eric C. Greene13: B direction of buffer flow ▼ set #1 Atto56S-dsDNAs ▼ set #2 Atto565-dsDNAs single 7,249-nt unit of M13mp18 _L_ ssDNA biotin \. 1_IL 2 3 4 3._t 5 extended loop (not monitored) _/_ -I- \ anchor 12 pm (-36,245 nt); Rad51/RecA (plus flow, L= 18 pm) How to study genome stability maintenance? Step6: study the molecular mechanisms by the means of single-molecule techniques. DNA RECOMBINATION Base triplet stepping by the Rad51/RecA family of recombinases Ja YD Lee,1 Tsuyoshi Terakawa,12* Zhi Qj,1* Justin B. Steinfeld,1 Sy Redding,1 YoungIIo Kwon,4 William A. Gaines,4 Weiring Zhao,4 Patrick Sung,4 Eric C. Greene13: B direction of buffer flow ▼ set #1 Atto56S-dsDNAs ▼ set #2 Atto565-dsDNAs single 7,249-nt unit of M13mp18 _Ĺ_ ssDNA biotin \. i—1_ 2 3 4 3._I_ 5 extended loop (not monitored) _l_ -I- 'mmmmmmmmmmmmmmmmk 7 barrier NPid bilayer fused silica slide 12 pm (-36,245 nt); Rad51/RecA (plus flow, L = 18 pm) \ anchor fused silica slide Rad51/RecA ssDNA anchors 40 How to study genome stability maintenance? Step6: study the molecular mechanisms by the means of single-molecule techniques. DNA RECOMBINATION Base triplet stepping by the Rad51/RecA family of recombinases Ja YU Lee,1 Tsuyoshl Terakawa,1-1* Zhi Qi,1* Justin B. Steinfeld,1 Sy Redding,3! YonngHo Kwon* William A. Gaines,* Webdng Zhao,* Patrick Sung,* Eric C. Greene' 5t Roc A RecAJ RecA' 5' 2 JRecA' 3 I (triplet 1) RecA1 (triplet5) B v c K C C 01 rr 1 1 1 II II 1 \ +* 4 11# M 111 4 \ *>* 4 \ 123 123123 123 Nucleotide position inject dsDNA 41 Transient summary IV 42 Transient summary IV There are different techniques that allow us understand any given pathway 42 Transient summary IV There are different techniques that allow us understand any given pathway The techniques must be combined, in order to get a full picture of the pathway Transient summary IV There are different techniques that allow us understand any given pathway The techniques must be combined, in order to get a full picture of the pathway Use whatever technique at hand that will help you answer your scientific question Summary 43 Summary Maintenance of genome stability is a complex endeavour, which requires intricate interplay of multiple pathways 43 Summary Maintenance of genome stability is a complex endeavour, which requires intricate interplay of multiple pathways Cells use sophisticated mechanisms in deciding which pathway to use at any given moment 43 Summary Maintenance of genome stability is a complex endeavour, which requires intricate interplay of multiple pathways Cells use sophisticated mechanisms in deciding which pathway to use at any given moment Majority of factors responsible for maintaining genome stability acts in complexes, let those be dynamic or not 43 44