MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 9 • DNA repair mechanism and recombination. 2 Department of Experimental Biology muni SCI Mutagenecity = Carcinogenesis • Ames tests thousands of chemicals and creates a catalog of substances, increasing the frequency of mutations. • Thus immeasurable becomes measurable. • Important observation was made: mutagenic substances have tendency to be carcinogens. • Also works for X-rays, tobacco smoke. 3 Department of Experimental Biology muni SCI Genetic stability depends on the DNA repair mechanisms • DNA repair is a collection of cellular responses by which a cell identifies and corrects any damage to the DNA molecules that encode its genome. • The frequency of mutations is kept to a minimum in healthy individuals. • Extremely accurate DNA replication and existence of DNA-repair mechanisms contributes to it. • Most correction mechanisms use the existence of two copies of genetic information in the double helix of DNA. • The damaged strand is identified by atypical structures and is repaired according to undamaged strand. Mutations in genes encoding proteins involved in DNA-repair increase the frequency of mutations, often lead to a predisposition to cancer. muni 4 Department of Experimental Biology D U 1 DNA repair For most repair mechanisms, there are three stages: • Identification of the damaged DNA section and its removal. o Nucleases split covalent bonds between damaged nucleotides and the rest of the DNA strand, o The gap is created on one strand. • Repair DNA-polymerase is attached to the 3-OH end of broken strand and starts polymerization according to intact strand. o Filling the gap. • Sugar-phosphate skeleton is healed by DNA-ligase. 5 Department of Experimental Biology muni SCI Stages of DNA-repair 5' 5' 3' Normal DNA i i i i i I I I i i I j, i i i i i i i i i i i Damadfed DNA I I I I \W\ I I I I ........... 5' 3' 5' 3' 5' 3' Damaged DNA region removed with endonuclease QH P ttt ttt ........... DNA respred with Repair DNAx>lymerase i i I i i i i I i i i ........... In 'Nick' closed with DNA ligase I I I I III I I I I ........... 6 Department of Experimental Biology i SCI Types of DNA-repair Direct enzymatic repair • Restoration to the original state. • Without cleavage and resynthesis of DNA. o Photoreactivation. o Removal of methyl group - Dealkylation. Indirect • Excision repair, o Base. o Nucleotides. o Mismatch - controlled by methylation. • Recombination/post replication. Inducible • SOS-response. 7 Department of Experimental Biology ^Direct enzymatic repair or Damage Reversal jsingle strand Damage Repair 1 .Photoreactivation 2.Removal of methyl groups. 1. Base Excision Repair 2. Nucleotide excision Repair 3. Mismatch Repair >ouble strand Damage 1 .Homologous Recombination 2.Non-Homologous end joining 3.SOS Response muni SCI Types of DNA-repair DNA damaging agents DNA damage types DNA repair mechanisms DNA replication stress If Base mismatches HJI ML Oxygen radicals O'H Ionizing radiation v# Polyaromatic hydrocarbons Ionizing radiation Chemotherapeutics $ UV light-j§j- Chemotherapeutics $ If If If vrrnKfTRKwr ssDNA breaks DNA adducts Abasie sites dsDNA breaks Intrastrand crosslinks Interstrand crosslinks Insertions/deletions 8-Oxoguanine A, K3 I I Mismatch repair Base-excision repair Nucleotide-excision repair dsDNA break repair 1 i Transcription-coupled/ Homologous recombination/ global genome repair non-homologous end-joining muni 8 Department of Experimental Biology https://blog.crownbio.com/dna-damage-response O 0 J. Mechanism of DNA-repairs • Enzymes search for damaged DNA sites and activate repair. Genetic apparatus for DNA repair: • Very conservative, used by bacteria and humans. • About 100 genes. • Activates various mechanisms that can interconnect. 9 Department of Experimental Biology muni SCI The most common types of DNA damage • Depurination - removal of purine bases in DNA without disruption of the sugar-phosphate skeleton (the most common spontaneous mutation, 100 times more common than depyrimidination). Depurination prevents replication and transcription. • Deamination - loss of the amino group of cytosine to form uracil. (A) DEPURINATION 0 1 0=P-0-CH2 N N H GUANINE H,0 N N GUANINE depurinated sugar (B) DEAMINATION CYTOSINE HI N 0=P—O —CH, I O 10 Department of Experimental Biology DNA strand Figure 6-23 Essential Cell Biology 3/e (C Garland Science 2010) H20 1 T NH, URACIL O N 0=P—O — CH, I O DNA strand muni SCI The most common types of DNA damage (a) • Wrong base pairing - "mismatch,,. • Covalent connection of adjacent pyrimidines, consequence of UV radiation: • Dimers thymine-thymine joined by aromatic (cyclobutane) circle. • Less often appear dimers thymine -cytosine or cytosine - cytosine. 11 Department of Experimental Biology Reactive double bonds - (b) Reactive groups Adjacent thymine residues Pyrimidine residues linked between carbon atoms 5 and 6 of each ring Sugar-phosphate backbone UV light Pyrimidine residues linked between carbon atom 6 of thymine and carbon atom 4 of cytosine UV light Adjacent thymine (5') and cytosine (3') residues Distorted sugar-phosphate backbone muni SCI UV radiation - physical mutagens After absorption of UV, pyrimidines become ^2 very reactive and change themself to A H 1^ / pyrimidine hydrates and then to the dimers. a+H0 N" M 1 > H Pyrimidine dimers results in formation of the I I replication fork and block of replication. „ x . „ ± . u ± Cytosine Cytosine hydrate H ^-c CH3 H3C, I II + S!l^ H H" Thymine ? Thymine UV CH3 CH3 H. H O N H r H I Thymine dimer 12 Define footer - presentation title / department Thymine dimers induced by UV light. j u n i SCI Photoreactivation - direct repair 5' 3' 5' 3'1 C C T T A Thymine dimer 51; 31: 5'j 3' 3' I 5' UV irradiation T = T 3' I 5' binding of photolyase to photolyase thymine dimer T = T cleavage of crosslinks 3' 5' I removal of enzyme after cleavage www.thevirtualnotebook.com •1. Photoreactivation is a direct enzymatic cleavage of thymine dimers activated by visible light. • Present in prokaryotes (e.g. E.coli) and plants exposed to sunlight. • In mammals replaced by excision repair. • Daylight activates the enzyme and thus ensures cleavage of covalent cross-bond. • Binding to dimers occurs even in the dark. 13 Department of Experimental Biology https://www.thevirtualnotebook.com/dna-repair-definition-mechanism-types/ muni SCI Photoreactivation - direct repair • Mechanism • Enzyme photolyase (encoded by phr gene) binds to a pyrimidine dimer. • Visible light (345 - 400 nm) shines on cell then FADH absorbs that light and release electron. • Electron interact with dimer. • Then splitting of cyclobutane ring in dimer due to electron interaction. • Finally, enzyme leaves the DNA and the DNA structure returned to its prior state - the result is monomerization. 14 Department of Experimental Biology https://www.easybiologyclass.com/photoreactivation-dna-repair-uv-induced-dna-damages/ Photo reactivation - direct repair Visible light o o sun UVR /WW MTHF/FMN Photochemical T reaction Formation of pyrimidine dimer in LiV-cxpo&ed DNA si Photolyase DNA-photolyase complex Restoration of normal base pair 15 Department of Experimental Biology Journal of Nucleic Acids, January 2010, (6551):592980 • The pyrimidine lesion, thymine dimers, are repaired by photoreactivating enzyme "photolyase". • The light energy (>380 nm) is trapped by the antenna molecules of photolyase (such as MTHF/8-HDF/FMN) and transfers them to catalytic cofactor FADH- which becomes excited. • FADH- transfers energy to the pyrimidine dimer and splits them into two monomeric unit, and then electron is transferred back to the flavin molecule. muni SCI Repair of alkylated DNA Direct repair - dealkylation - adaptive response. The enzyme: alkyltransferase (demethylase) (06-metylguanin-DNA-metyltransferase = Ada-protein). Alkyltransferase takes the methyl group from methylated guanine (the acceptor is cysteine Ada-protein) or from phosphate groups of sugar-phosphate backbone. Active Cys — SH Inactive Cys — SH-CU,} OCH.; N N > H2N^N^N Mi.tlivlíríiivforjwi H2N \ R 16 Department of Experimental Biology 0G-Methyl£uanine nucleotide Guanine nucleotide muni SCI Repair of alkylated DNA Ada-protein is a bifunctional protein and uses a N-terminal Cys38 residue to remove a methyl group from Sp-methylphosphotriester and a C-terminal Cys321 residue to remove a methyl adduct from 06-methylguanaine. Upon receiving a methyl group from the damaged DNA, Ada turns into a transcriptional activator and activates its own expression and transcription of genes repairing damaged caused by of alkylation, AlkB. o;er ada. alkB. ulkA and aidB \ Mclhylation Activate methyiation resistance genes V 5-ONA— f/ "b-ONA-ar Sp-methyl phosphotriester ( C-Ada § { 321 H3CN ^N-^N^NH2 DNA 0*-methyfguanine 17 Department of Experimental Biology Journal of Inorganic Biochemistry, Volume 100, Issue 4, April 2006, Pages 670-678. muni SCI Repair of alkylated DNA Ad;) protein Ada protein V tormina I domain CH] from phosphodiester DNA backbone from methylated base Ada converts to a transcriptional activator Protein degradation C-icrminal domain 1 Protein ^^^^^^ ^^^^^ ICHjl degradation Transcriptional activation Transcriptional activation ada \ I Repair Repair P04.CH3 ^meT Defense against SN1 type agents / Defense against SN2 type agents alkB alkA aidB —SM ■ \ \ \ Repair Repair Inactivation ImcA 3meA, 3meG of MNNG 3meC tf2meT, 02meC I 7meG, 7meA I Defense against SN1 type agents Defense against SN1 & SN2 type agents 18 Department of Experimental Biology Direct DNA Lesion Reversal and Excision Repair in Escherichia coli, SOPHIE COUVE et al., February 19 , 2013. muni SCI Excision repair mechanism • Excision repair is a general mechanism of DNA repair. • Various enzymes are involved that can sense DNA damage. • During excision repair bases and nucleotides are removed from damaged strands. Gap is then patched using complementarity with the remaining strand. • Excision repair is broadly categorized into: o Base excision repair. o Nucleotide excision repair, o Mismatch repair. 19 Department of Experimental Biology muni SCI Excision repair mechanism Repair mechanisms that include nucleotide removal utilize a common four-step pathway: 1. Detection: The damaged section of the DNA is recognized. 2. Excision: DNA-repair endonucleases nick the phosphodiester backbone on one or both sides of the DNA damage and remove one or more nucleotides. Polymerization: DNA polymerase adds nucleotides to the newly exposed 3'-OH group by using the other strand as a template. Ligation: DNA ligase seals the nicks in the sugar-phosphate backbone. Repair — endonuclease 5' 3' ft -7 Damage in one strand of DNA 3' 5' 5' 3' 5' 3' 5' 3' Q Excision of damaged DNA .3' 5'i 3' 5' DNA polymerase Q DNA polymerization to fill gap 3' 5' DNA ligase Q Remaining nick sealed by DNA ligase 3' 5' " 2Q12PBVKF1 Education Ire Department of Experimental Biology https://www.thevirtualnotebook.com/dna-repair-definition-mechanism-types/ muni SCI Base excision repair (BER) pathway • Base excision DNA repair pathways remove abnormal or chemically modified bases from DNA. • Base from the nucleotide within DNA can be removed in several ways: o either by direct action of an agent such as radiation, o or by spontaneous hydrolysis, o by an attack of oxygen free radicals, or o by DNAglycosylases. • BER repairs DNA bases damaged by: o Alkylation. o Deamination. o Oxidation. o Lost of base - Depurination, Depyrimidation. 21 Department of Experimental Biology muni SCI Base excision repair (BER) pathway • In BER pathway, a modified base is first excised and then the entire nucleotide is replaced. • The excision of modified bases is catalyzed by a set of enzymes called DNA glycosylases. • Each of which recognizes and removes a specific type of modified base by cleaving the bond that links that base to the 1'-carbon atom of deoxyribose sugar. • Each glycosylase recognizes a specific type of altered base, such as deaminated bases, oxidized bases, and so on. 22 Department of Experimental Biology https://www.thevirtualnotebook.com/dna-repair-definition-mechanism-types/ muni SCI Base excision repair (BER) mechanism S' HHHHHHHHHHHHK 3' 3 5 • The glycosylases cleave the glycosidic bond between Cytosine Deamination \J J J \J J ......*....... the abnormal base and 2-deoxyribose, creating apurinic or apyrimidinic sites (AP sites) with missing DNA glycosylase binds I baSOS. to uracil glycosylase 3' —s • An AP endonuclease then senses the minor distortion Excisionofuraciuu, j flPsite 0f the DNA double helix and initiates excision of the 5'^^^^^^^^^3' single AP nucleotide. Phosphodiesterase removes 7 : mlBing' : 5' sugar-phosphate bond. phosphodiesterase J /„uc|eotide removes sugar-phosphate* X l^^^^^^^^^^l • DNA polymerase then replaces the missing nucleotide 3 DNApolymerase I 5 according to the specifications of the complementary 5 \/b'"\ 3 strand. | siteo»repair . DNA ligase seals the nick. 5' ■■■■■■■■■■■■■ 3' www.thevirtualnotebook.com |^| 23 Department of Experimental Biology https://www.thevirtualnotebook.com/dna-repair-definition-mechanism-types/ O 0 J. Base excision repair (BER) mechanism • BER can remove • Short-patch -nucleotide. • Long-patch - couple of nucleotides. 24 Department of Experimental Biology NEIL1-3. PNKP 5'rmm 3" ,.999999 5. ♦ «"^ DNA glycosylase 5 6 i ' A A A 3" 3,2_2_2_2_2_£ 5- Damaged DNA base (♦) Newly inserted DNA base (O) 3. 9 9 9 9 9 9 5. Pol 5/e '3- 9 9 9 9 9 9 5 4 Pol p FEN-1/PCNA 5 TT" %TT3' v 2 2 9 9 9 9 5. 1 Pol p .-9 9 9 9 9 9 5. I XRCC1-Lig Ilia 3' 5' urn%3' r 9 9 9 9 9 9 5. I Lig l/PCNA 5 ó ó 6 ó ó Ó 3 Long-patch BER 6 6 6 6 6 6 ,■9 9 9 9 9 9 5. Short-patch BER Molecular and Cellular Biology, Volume 36, Issue 10, 15 May 2016, Pages 1426-1437. i SCI Nucleotide excision repair (NER) pathway • Nucleotide excision DNA repair pathways remove larger defects like thymine dimers and bases with bulky side-groups from DNA. • NER is perhaps the most flexible mechanism in terms of the diversity of lesions that are recognized and repaired. • The most significant of these lesions are pyrimidine dimers induced by UV light (cyclobutane pyrimidine dimers and 6-4 photoproducts), and other NER substrates include bulky chemical adducts, DNA intra-strand crosslinks, and some forms of oxidative damage. • These types of lesions cause both a helical distortion of the DNA duplex and a modification of the DNA chemistry, both of which are hallmark features recognized by NER. https://blog.crownbio.com/dna-damage-response IVI U 111 25 Department of Experimental Biology _ _ T https://www.thevirtualnotebook.com/dna-repair-definition-mechanism-types/ V. f Nucleotide excision repair (NER) pathway • While mechanistically similar to BER, the NER pathway is more complex, requiring some thirty different proteins to carry out a multi-step 'cut-and-patch'-like mechanism. • The defects in NER cause several human genetic disorders, including: o Xeroderma pigmentosum o Cockayne syndrome o Trichothiodystrophy • These all are characterized by extreme sun sensitivity. • In addition, these diseases demonstrate overlapping symptoms associated with cancer, developmental delay, immunological defects, neurodegeneration, and premature aging. muni 26 Department of Experimental Biology r> r» t Nucleotide excision repair (NER) mechanism • Thymine dimer is repaired by NER. • Damaged nucleotide(s) are removed along with a surrounding patch of DNA. • In this process, a helicase (DNA-opening enzyme) cranks open the DNA to form a bubble. • DNA-cutting enzymes chop out the damaged part of the bubble. • A DNA polymerase replaces the missing DNA, and a DNA ligase seals the gap in the backbone of the strand. 5' 3' UUUULP ' UUUUU UV mention oroíuw- III III II III III ..... Ill III II uv raaiaTlon produces , no_nnwfA_nnn athvminedimer' 3' 5' 5' LTD nn li 3' Once the dimer has been f,nn nnwa nnn_ detected, the surrounding DNA is opened to form a bubble. I ,cn6 'off3' Enzymes cut the damaged region 1..... "i, 11 out of the bubble. nn_nn^ra nnn 3' 5' 3' [ [""t j [ 3 A DNA polymerase replaces III in II III in li II ll III III III II the excised (cut-out) DNA, and A t\_ a ligase seals the backbone. □ 5' 27 Department of Experimental Biology https://www.khanacademy.org/science/biology/dna-as-the-genetic-material/dna-replication/a/dna-proofreading-and-repair muni SCI Nucleotide excision repair (NER) in bacteria TO535Söaei*ö8SSS355 9999999999999999999999 -A UvrA C UvrA uvrBC recruitment uvrA release UvrC UvrB 9999999999999999999999 J Incision UvrB "AAAAAŽAAAAAÍiAAg5^555 9999999999999999999999 UvrD-mediated fragment release and release ol UvrBC S533J' 9999999999999999999999 I Polymerization and ligation .A ' DNA JLigase J "J333aA'fíSAAA'ííAAb^I53 9999999999999999999999 28 Department of Experimental Biology • UvrABC endonuclease is a complex in bacteria involved NER, is sometimes called an excinuclease. ABC excinuclease - subunits encoded by uvrA, uvrB and uvrC genes. It moves along DNA and detects Thymine dimers for excision endonuclease. • UvrA and UvrB complex attach on distortion site then UvrA will dissociates. • UvrB attracts UvrC and nicks 5 nucleotides at 3' side of DNA while 8 nucleotides nicks at 5' side of DNA will be produced by UvrC subunit. • UvrD (DNA helicase II) removes 12 oligonucleotides. • DNA polymerase I now fills in gap in 5' —► 3' direction. DNA ligase seals the gaps. Current Opinion in Microbiology, Volume 4, Issue 2, 1 April 2001, Pages 178-185 muni SCI Nucleotide excision repair (NER) in eukaryotes • Bulky DNA lesions that destabilize duplex DNA. • Strongly distorting lesions are directly recognized by XPC-RAD23B, which binds the nondamaged strand opposite the lesion. • TFIIH interacts with XPC-RAD23B and open DNA with RPA • The XPF recruitment makes incision 5' to the lesion. • Initiation of repair synthesis by Pol 5 and Pol k or Pol z and, followed by 3' incision by XPG. • Sealing of the nick by DNA ligase llla/XRCC1 orDNAIigase I completes the process. 29 Department of Experimental Biology •'mini ^ i i i i i i ** 3 i ii i i i * a i i i i i i ft- H Initial damage I recognition Operwrvg of UNA double helix •Ad XPG •ndonuclMMi xp-e cut xp-g on "TT** *'TTTr i.....1 * *......I DNA polymerase DNA ligase * i i i i i i T T i i i i i i 3 r i i i i 11 a a 11 i i i 1 s-Wild type DNA muni SCI o o t Š s o a. O Nucleotide excision repair (NER) in eukaryotes GG-NER TC-NER UV UV-DDB / UV CSA CSB ý RNApol2 RAD23B XPC ERCC1/XPF /-riiiiiiiiiiiiiiiiiiUiTx XPG RPA 1 DNA polymerases 6^ PCNA^ • NER is executed by two different damage-detection mechanisms, which utilize the same machinery to excise and repair the damage. • Transcription-coupled NER (TC-NER) is initiated by stalling of RNA polymerase 2 on a lesion present in the transcribed strand of active genes, and depends on recruitment of the CSA and CSB proteins. • Other lesions are removed by global genome NER (GG-NER), which is initiated by the UV-DDB ubiquitin ligase complex and the heterotrimericXPC/RAD23/CETN2 complex. • Following detection, the TFIIH complex is recruited, and unwinds a stretch of approximately 30 nucleotides around the damage, providing access for other repair factors. 30 Department of Experimental Biology Epigenetics & Chromatin, January 2012, 5(1):4 muni SCI BER and NER comparison in eukaryotes ROS Recognition Excision Resynthesis VVvl, Non-bulky lesion i i i i i i i ....... 4 1111111 11111111 ......■......... vAPEl i i i i i i i i i i i i i i i ................ A Bulky lesion i i i i i i tUA i i i i I i .......VI....... UV-DDB- (DDB1\ VddbÍNL 1 1 1 1 mm .1 jj u ' XPC M XPF 111'' inj.^yy^ , li 1111111 Long-patch (2-~13 nt) BER Short-patch (lnt) • DNA lesions: • non-bulky (non-helix distorting) • bulky (helix distorting) lesions. • In human cells, BER and NER are responsible for the removal of non-bulky and bulky DNA lesions, respectively. Excision product (22-32 nt) 31 Department of Experimental Biology https://encyclopedia.pub/entry/176 i SCI Mismatch repair (MMR) pathways • A system of recognizing and repairing errors that occur during DNA replication and/or repairing errors caused by DNA damage. • There are many different types of mismatch repair systems; however, mismatch repair mechanisms are strand-specific. • Accounts for 99% of all repairs. I C T C A first round replication (mis-incorporation) second round replication 1 32 Department of Experimental Biology https://slideplayer.com/slide/15878337/ JUT JJJ muni SCI Mismatch repair (MMR) pathways Main challenges mismatch repair proteins face: 1. Must scan the genome for mismatches (sensed as a distortion of the double helix). 2. Must repair before the next round of replication. 3. Must repair the "mutant" nucleotide on the newly synthesized strand. 33 Department of Experimental Biology https://slideplayer.com/slide/15878337/ muni SCI Mismatch repair (MMR) pathways in prokaryotes MutH- Is* ADP 3'I 5M T 3'C mismatch repaired • MMR removes single base pair errors that escape proofreading. Well studied in E. coli. Proteins are called the Mut proteins: • MutS - recognizes mismatches. • MutH - nicks one strand ahead of the mutation. • UvrD, DNA-helicase II, and exonuclease are recruited to remove base pairs including the mismatch. • DNA Pol III fills in the gap and ligase sealed it. 34 Department of Experimental Biology https://slideplayer.com/slide/15878337/ muni SCI Mismatch repair (MMR) pathways in prokaryotes How does the Mismatch Repair System "know" which strand has the mutation? It is because the newly synthesized strand of DNA is not methylated by the enzyme DNA methyltransferase, DAM. MutH nick MutH does nicks the newly synthesized DNA strand. 35 Department of Experimental Biology https://slideplayer.com/slide/15878337/ muni SCI DNA methylation during replication in prokaryotes When DNA is replicated, the old strand is methylated, thus the DNA double helix, as a whole, is "hemi-methylated", but there is a delay in methylating the new strand and. CH3 G AT C C TAG CH3 CCTGG GGACC CH3 CH I REPLICATION OF DNA Methylase works with a delay of 2 - 3 minutes. During this period, parent chains can be distinguished from newly synthesized. Old strand CH3 GGACC New strand TWO MOLECULES OF HEMI-METHYLATED DNA CCTGG GGACC CH, 36 Define footer - presentation title / department https://www.sciencedirect.com/topics/nursing-and-health-professions/dna-adenine-methyltransferase muni SCI DNA methylation during replication in prokaryotes • Methylation is usually subject to adenine or cytosine, provided by enzymes -methylases. • In bacteria eg. Dam-methylase methylates adenine in the GATC sequence. G AT C CCTGG CH3 CH3 muni 37 Department of Experimental Biology r> r» t Mismatch repair (MMR) pathways in eukaryotes • In eukaryotes the MutS Homologs (MSH) were discovered by analyzing for similarities in protein sequences. • In eukaryotes the template DNA is not methylated. • MMR in eukaryotes is coupled with DNA replication. • The interaction of MutLa, member of MMR repair complex with PCNA provides the strand-discrimination signal for MMR, because RFC asymmetrically loads PCNA onto DNA at a nick. • Mutations in MSH genes result in several malignant diseases, such as hereditary nonpolyposis colorectral cancers. 38 Department of Experimental Biology muni SCI Mismatch repair mechanism in eukaryotes • The majority of replication errors are corrected by MMR that depends on DNA ends made by MutLa (M1), ends at Okazaki's fragments (M2) and RNaseH2 generated ends (M3). • The M1 mechanism is the most important and M3 being the least important. 5' RNA primer © Ribonucleotide ♦ Mismatch M] = Processing ends made by MutLa Mj = Processing 5' - ends during Okazaki fragment maturation M3 = Processing ends generated by RNase H2 (and others?) o 3' .Vi M 39 Department of Experimental Biology Annu Rev Genet. 2015; 49: 291-313. muni SCI Mismatch repair mechanism in eukaryotes Factors required for mammalian DNA mismatch repair _*s pathway: • Primary mismatch recognition factor MutSa (MSH2- MutLa J MSH6 heterodimer). Mismatch PCNA e • MutLa endonuclease (MLH1-PMS2 heterodimer). • Replicative clamp PCNA. • The clamp loader RFC. • The 5'—»3' exonuclease EX01. DNA polymerase 5 (Pol 5). \~ Po,5>PCNA RFC.Iigasel • DNA Ligase I. 40 Department of Experimental Biology Science China. Life sciences, October, 2017, 60(10) muni SCI Mismatch repair mechanism in eukaryotes PCNA Incision by MutL 5' MutS« i Excision lEXOt-dopondont by EX01 / \ strind dlsP ndontMMR)/ \ by Fol A J \ (EXOMn RPA displacement synthesis holoentyme (HEI indcpundonl MMR) Gap filling by Pol b or Pol i HE 41 Department of Experimental Biology The MutSa recognizes the mismatch and cooperates with loaded PCNA to activate MutLa endonuclease. The activated MutLa endonuclease incises the discontinuous daughter strand 5' and 3' to the mismatch An incision produced by MutLa 5' to the mismatch is utilized by MutSa-activated EX01 to enter the DNA and remove the mismatch in a 5'—>3' excision reaction modulated by RPA. The generated gap is repaired by the Pol 5 holoenzyme. https://biochem.siu.edu/faculty/Kadyrov.html muni SCI Double-srand DNA brakes • A double-strand DNA break (DSB) occurs or arises when both strands of the DNA duplex are severed, often as the result of ionizing radiation. • DSB can also be caused by mechanical stress on chromosomes or when a replicative DNA polymerase encounters a DNA single-strand break or other type of DNA lesion. • They are particularly deleterious as the DNA ends can promote potentially gene loss or lethal chromosomal rearrangements. • Can be repaired by: • Homologous Recombination (HR). • Non-Homologous-End Joining (NHEJ). • Microhomology-Mediated-End-Joining (MMEJ). 42 Department of Experimental Biology https://www.nature.com/subjects/double-strand-dna-breaks muni SCI Double-srand DNA brakes Ionising Radiation Induced Damage Rapid repair Slow repair Very slow repair Prone to small errors Highly accurate Prone to significant alterations Active throughout cell cycle Active in S and G2 phases Fall-back pathway Extensively modelled Limited modelling Limited modellrng 43 Department of Experimental Biology Cancers, February, 2019, 11(2):205 muni SCI Double-srand DNA brakes • DSBs can be repaired by several different mechanisms: • nonhomologous end joining -NHEJ • microhomology-mediated end joining - MMEJ • double Holliday junction -dHJ • double-strand break repair-DSBR • synthesis-dependent strand annealing - SDSA • break-induced replication -BIR • single-strand annealing -SSA. SSA 1 Extensive resection 1 Donor independent > Loss of repeat and intervening DNA Pathways of DSB Repair NHEJ MMEJ/aEJ ♦ Homologous Recombination i D-loop Resection Donor dependent High fideltiy • No or little resection • Donor independent • Variable fidelity dHJ ...........r»l Non-crossover Non-crossover BIR SDSA Crossover Non-crossover Non-crossover dHJ Resolution dHJ Dissolution 44 Department of Experimental Biology https://microbiology.ucdavis.edu/heyer/wordpress/research muni SCI Homologous recombination (HR) • HR repair DNA breaks by retrieving sequence information from undamaged DNA. • DSB are repaired mostly by HR and NHEJ pathways. (A) NONHOMOLOGOUS END JOINING (B) HOMOLOGOUS RECOMBINATION accidental double-strand break loss of nucleotides due to degradation from ends end joining 45 Department of Experimental Biology deletion of DNA sequence Figure 5-51 Molecular Biology of the Cell 5/e I© Garland Science 2008) I-1 isister chromatids loss of nucleotides due to degradation from ends end processing and homologous recombination damage repaired accurately using information from sister chromatid muni SCI Model of homologous recombination Gene 5' * 3'5' Gene B 3' 3'' 5'3 '5' (T) A double-strand break in one of two homologs is converted to a double-strand gap by the action of exonucleases. Strands with 3'ends are degraded less than those with 5'ends, producing 3' single-strand extensions. (D The invading 3'end is extended by DNA polymerase plus branch migration, eventually generating a DNA molecule with two crossovers called Holliday >lr- intermediates. (2) An exposed 3'end pairs with its complement in the intact homolog. The other strand of the duplex is displaced. /DCZ I @ Further DNA replication replaces the DNA missing from the site of the original double-strand break. (5) Cleavage of the Holliday intermediates by specialized nucleases generates either of the two recombination products. In product set 2, the DNA on either side of the region undergoing repair is recombined. Product set 1 Product set 2 46 Department of Experimental Biology muni SCI Model of homologous recombination Strand invasion (strand exchange) is a key step in homologous recombination 47 Department of Experimental Biology aligned homologous duplexes 5' 3' A a B b C c single-strand breaks in each duplex top duplex — hybrid duplexes muni SCI Copyright ffl 2008 Pearson Education, Inc., publishing as Pearson Benjamin Currimings. Model of homologous recombination Resolving Holliday junctions is a key step (final step) to finishing genetic exchange. 48 Department of Experimental Biology "patch" or noncrossover products no reassortment _a_s_£_ "splice" or crossover products reassortment of flanking genes A B r 5 9 A b c 3"<-' C 5*i— a b C a b c Copyrighi©2008 Pearson Education. Inc.. pubiisning as Pearson Beniamm Cummrngs muni SCI Model of homologous recombination The double-strand break-repair model describes many recombination events. 49 Department of Experimental Biology second strand invasion and DNA repair synthesis from 3' ends branch migration and formation of an intermediate with two Holliday junctions a b Copyright © 2008 Pearson Education, Inc.. publishing as Pearson Benjamin Cummings. muni SCI Model of homologous recombination • Prokaryotic and eukaryotic factors catalyzing recombination steps. Recombination Step E. coli Protein Catalyst Eukaryotic Protein Catalyst Pairing homologous DNAs and strand invasion Introduction of DSB Processing DNA breaks to generate single strands for invasion Assembly of strand-exchange proteins Holliday junction recognition and branch migration Resolution of Holliday junctions RecA protein None RecBCD helicase/nuclease RecBCD and RecFOR RuvAB complex RuvC Rad51 Dcm1 {in meiosis) Spo11 (in meiosis) HO (for mating-type switching) MRX protein (also called Rad50/58/60 nuclease) Rad52 and Rad59 Unknown Perhaps Rad51c-XRCC3 complex and others CopynflW O 2006 Pearson Education, inc.. publishing a5 Pearson Benjamin CumrrnnpA 50 Department of Experimental Biology muni SCI Prokaryotic homologous recombination • The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. • The RecBCD enzyme is, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. • When Chi is in the RecC tunnel, o RecC signals RecD to stop unwinding DNA; o RecD signals RecB to nick the DNA and to begin loading RecA. • This enzyme is a prototypical example of a molecular machine: several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. 2. Hi cC signals RecD 10 slop 3. When slopped, RecD signals RccB to cut DNA nuclease and RecA-ioading 4. RccB cuts whore it is and continues unwinding DNA, loading RecA 51 Department of Experimental Biology June 2020Nucleic Acids Research 48(14) Microbiol Mol Biol Rev. 2008 Dec; 72(4): 642-671 muni SCI 9 Prokaryotic homologous recombination model ran —o- RecA I 3' © Reciprocal /" break-join ^ \ Non-reciprocal * break-copy (BIR) 5 *-aw\A>« AAAAAA* 52 Department of Experimental Biology RecBCD binds a ds DNA end (a) and unwinds the DNA, producing loop-tail structures (b) that are converted into twin-loop structures (c) by annealing of the tails. At Chi, RecBCD nicks the 3'-ended strand (d) and loads RecA (e); later, the RecBCD subunits disassemble. The ssDNA-RecA filament invades intact homologous DNA to form a D-loop (f) D-loop is converted into a Holliday junction and resolved into reciprocal recombinants (g). Alternatively, the 3'-end in the D-loop prime synthesis of DNA and generate a non-reciprocal recombinant (h). Scientific Reports volume 10, Article number: 19415 (2020) muni SCI Prokaryotic homologous recombination model • Chi sites controls RecBCD activity and relative recombination frequency. • y-site 5'-GCTGGTGG-3'. donor DNA 5'a (linear) 34.Radb4B.Rdh54. Hop2-Mndl) IIIIIIIIIIIIIHIIIIIIIIIIIII The recombinases Rad51 or Dmc1 (green circles) assemble onto the ssDNA tails that form a helical protein filament, which is known as the presynaptic filament. The presynaptic filament binds duplex DNAto form the synaptic complex and 'searches' for DNA homology in the duplex DNA molecule. With function of Rad54, Rad54B, Rdh54 and Hop2-Mnd1. ssDNA invades the homologous region in the duplex to form a DNA joint, known as the displacement (D)-loop. 64 Department of Experimental Biology Molecular cell biology, October 2006, volume 7, 739. muni SCI Eukaryotic HR pre-synapsis phase Human DMC1 can form either stacked rings or helical filaments on DNA, depending on whether ATP is present or not. DMCl ssDNA ' Left electron micrograph shows helical filaments of DMC1 protein on ssDNA. These filaments are the catalytically active form of DMCL Right electron micrograph shows stacked DMC1 rings (two such stacked rings are indicated by the double arrow) on ssDNA. These stacked DMC1 rings seem incapable of mediating the HR reaction. - ATP . at r b DMCl Bbmanta recombinase active c DMCl rings: recombinase inactive y) nm 65 Department of Experimental Biology Molecular cell biology, October 2006, volume 7, 739. ■)Unm muni SCI Non-homologous end-joining (NHEJ) pathway • Non-homologous end-joining is a pathway that repairs double-strand breaks in DNA. • We call it "non-homologous" because the break ends are directly joined without the need for a homologous template. • This pathway often occurs when the cell is in G1 and a sister chromatid is not available for repair through homologous recombination. • Nonhomologous end joining uses proteins that recognize the broken ends of DNA, bind to the ends, and then joins them together. • Nonhomologous end joining is more error-prone than homologous recombination and often leads to deletions, insertions, and translocations (Various types of mutations). 66 Department of Experimental Biology https://www.thevirtualnotebook.com/dna-repair-definition-mechanism-types/ muni SCI Non-homologous end-joining (NHEJ) pathway HR utilizes a homologous stretch on a sister chromatid and single-strand overhangs are created. Formation of a joint molecule with the damaged and undamaged strands. DNA synthesis complete repair of the DSB. NHEJ brings the ends of the broken DNA molecule together by the formation of a synaptic complex, consisting of two DNA ends, two Ku70/80 and two DNA-PKCS molecules. Non-compatible DNA ends are processed to form ligatable termini, followed by repair of the break by the ligase IV/XRCC4 complex. B NuClecHyti processing ig \ 0-~> RPA Strand invasion ^-BRCA2 q—Ratf52 □ MA synthesis Resolution Department of Experimental Biology Cell Research volume 18, pagesl 14-124 (2008)Cite this article Binding ol KU7Q/3Q. DMA-PKcs Ku 70/80 Synaptic complex muni SCI Non-homologous end-joining (NHEJ) mechanism dm B DNA-PKcs ATM-mediated DMA-PKCs phosphorylation DNA-PKcs auiophosprioiyaiion Classical' processing □ SB subset processing Polymerase or Artemis XLF/ LigaselV Cern . XRCC4 □ \ Polymerase Li gase IV XRCC4 / A) The Ku70/80 heterodimer associates with the two ends of the broken DNA molecule. This DNA-Ku scaffold attracts DNA-PKCS, which protects the DNA termini against degradation and premature ligation. (B) The DNA-PKCS molecules on both DNA ends form a synaptic complex which tethers the DNA ends. Trans DNA-PKCS autophosphorylation then introduces a conformational change that makes the DNA termini accessible for other NHEJ enzymes. (C) Non-compatible DNA termini need to be processed before ligation can proceed. This can be done in the 'classical' way, by either filling (polymerases) or resection (Artemis) of single-strand overhangs. 68 Department of Experimental Biology Cell • (D) ligation of the blunted ends by ligase IV/XRCC4. Research, 2008, volume 18, pages 114-124. muni SCI Non-homologous end-joining (NHEJ) pathway Various non-homologous end joining pathways. * Blunt ends Ku binding ' y~ =0 e= =o>o= =:r^= ==o^o=jeo^b i b Incompatible 5' ends c Resection-dependent d Incompatible 3' ends * 3-phosphoglycolated compatible ends ends ^ I I I I End processing Ligation J^XRCC4 diml>r R&iection 34. /Synthesi R.e section 1 Í =0 €p=-=0= =C^=^) ^""=0 69 Department of Experimental Biology Nat Rev Mol Cell Biol. 2017 Aug; 18(8):495-506. muni SCI NHEJ in V(D)J recombination • Genes that encode immunoglobulins or T-cell receptors are not present in an active form in developing B- and T-lymphocytes, but need to be formed by the combination of gene segments. • This process is called V(D)J recombination. • Gene segments are classified into three groups: variable (V), diversity (D), and joining (J) segments. • In the case of an IgH gene, D and J segments are first joined, followed by the combination of the DJ assembly with a V segment. V regions 0 regions J regions Constant region i-i i-i i-1 i-i | D to J joining Annu. Rev. Biochem. 2021.90:137-164. muni 70 Department of Experimental Biology _ _ _ K Cell Research, 2008, volume 18, pages 114-124. OPT NHEJ in V(D)J recombination Gene segments are joined by the introduction of a DSB at the edges of selected segments by the RAG 1 and RAG 2 proteins, followed by removal of the intervening DNA and ligation of the segments. Before ligation the typical hairpin structure of the coding ends needs to be opened by the endonuclease Artemis. V(D)J recombination requires the NHEJ core enzymes (DNA-PKCS, Ku70/80, ligase IV, and XRCC4), indicating that ligation of the gene segments is mediated by the NHEJ process. s** ""V ends RAG 12 J_[ \ _ 71 Department of Experimental Biology Annu. Rev. Biochem. 2021.90:137-164. Cell Research, 2008, volume 18, pages 114-124. Artemis DNA-PKcs DNA-PKcs I Ligase IV KU70/80 /XRCC4 Coding joint NHEJ synaptic complex Hairpin opening Processing and joining Coding Joint ^^pj' lZZjj^I it! IndHs muni SCI DNA Strand break repair pathway choice End protection (resection usually <20nt) JL :5E = : 00 Ku7O-KuS0 Artemis DNA-PKcs Pol X. Pol u PAXX XLF XRCC4 DNA ligase IV \HEJ -. - End resection (> 20ntforSSA and >50 nt for HR) Sister chromatids (S-G2) PARPl \ Microhomology Pol« a-EJ BLM EXOl RPA Additional end resection Homology. RAD52 XPF-ERCC1 SSA BRCA1 BRCA2 RAD 5 4 HR 72 Department of Experimental Biology Nat Rev Mol Cell Biol. 2017 Aug; 18(8):495-506. muni SCI The Error-prone Repair System (SOS response) • The SOS repair is a complex set of processes that includes a bypass system that allows DNA replication to take place across pyrimidine dimers or other DNA distortions but at the cost of the fidelity of replication. • Emphatically, SOS repair is an error-prone system for DNA repair. • "SOS repair" refers to a cellular response to UV damage. • This error-prone repair system eliminates gaps in the newly synthesized strands opposite damaged nucleotides in the template strands but, in so doing, increases the frequency of replication errors. • The SOS-response has been found in many bacterial species, but not in eukaryotic cells. muni SCI The Error-prone Repair System (SOS response) NORMAL DNA LexA protein SOS BOX J SOS genes will not express inducer proteins SOS: SWITCHED OFF DAMAGED DNA LexA proteolysis by RecA SOS BOX SOS genes will be expressed to repair damaged DNA SOS SYSTEM: INACTIVATION AND ACTIVATION BIOLOGY -READER • SOS system consists of more than 40 genes and is regulated by the LexA repressor protein. These include the uvr genes needed for nucleotide excision repair and recA, which is involved in homologous recombination. • LexA binds to DNA sequence upstream of their coding region, called the SOS box. • The SOS response can also induce the expression of translesion polymerases encoded by the dinA, dinB and umuCD genes. 74 Department of Experimental Biology https://biologyreader.com/sos-repair.html https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Free_For_ AII_(Ahern_Rajagopal_and_Tan) muni SCI SOS response mechanism • In case of excessive DNA damage, cell responds by activating signal or RecA protein. A RecA protein specifically binds to the single stranded DNA. On binding with the single stranded DNA fragments, RecA forms a filament-like structure around the DNA. • Then, a LexA repressor comes in contact with the nucleoprotein filament assembled by the RecA protein. When RecA interacts with the repressor protein, it converts into RecA protease. RecA ť"N protease\_y RecA protein '-v— Damaged DNA RecA uvrA urnu A LexA repression Promoter of LexA Promoter of LexA 75 Department of Experimental Biology https://biologyreader.com/sos-repair.html muni SCI SOS response mechanism • The formation of RecA protease causes autocatalytic proteolysis of LexA repressor protein. Thus, a LexA protein could not bind with the SOS operator. • Inactivation of LexA protein activates the inducer proteins that repair the DNA damage but alters the DNA sequence. • After DNA repair, the RecA protein loses its efficiency to cause proteolysis, and the LexA protein will again bind to the SOS operator or switch off the SOS system. 76 Department of Experimental Biology https://biologyreader.com/sos-repair.html muni SCI Different repair mechanism for principal DNA lesions Alkylating Oxygen SponM wou* IUmj Kütmn v UV lieht , . 1 . Othťf máat r \ ran ^ agenti r.ulu.iK reaction errors Tl\ Cycluhutiint 0*-mine fl-oxopujninc thyminedimers in || O i + ♦ ttypaxanthirK' Inlriislrand crosslinks Mispaircd basts O i ' i v 1 N-a>eoxyguunosin-S- Ar- >a" «X pp phůUiproduCts XXX j? USA phn r L'WL'il O LHtccI repair 5jHitKlhyl S-hytlrDnynjetliyUiraLil phiwphoiricster .a. Abasw; iiies tí ■ L' l>J« P*' ra ťaf)y GiTUiíepaiís » r ^i,' H..OH vy I 4 UilťLl irp.m Bai« excision frjidir Nudcmidc rMisum repair AHernatrv rrpair Department of Experimental Biology J Nucleic Acids. 2010; 2010: 179594. IJoublr H ran J breaks CoJIapsod fork striiLlurcs Mismatch repair u Rti;i>mhinatii>n re [Mir muni SCI THANK YOU FOR YOUR ATTENTION 78 Department of Experimental Biology hffp://biocomica Is.bl09sp0i-.com http://biocomicals.blogspot.com/2011/05/dna-repair.html muni SCI