ENZYMES USED IM MOLECULAR BIOLOGY Nucleic acid processing enzymes •nucleases •polymerases •phosphatases •methylases •kinases •ligases •polynucleotidyl phosphorylase 2 Nucleases •hydrolases cleaving phosphodiester bonds •DNases, RNases, „non-specific“ 3 Nucleases •which phosphoester bond is hydrolyzed 4 3‘-OH, 5‘-phospho 5‘-OH, 3‘-phospho DNase I repair nucleases phosphodiesterase I S1, P1 nucleases DNase II phosphodiesterase II RNase A MNase Nucleases •endonuclease or exonucleases • • • • •exonucleases: –processive or distributive – – – – –direction of cleavage (5‘→3‘, 3‘→5‘) 5 association-(cleavage-translocation-cleavage)n (association-cleavage-dissociation)n Nucleases •other criteria (levels of selectivity/specificity): – –secondary structure of substrates (ss, ds, untwisted) – –preference for certain nucleotide (Pu vs Py or particular base) – RNases (A, U1, U2, T1, T2, PhyI, PhyII) – –sequence specificity (restriction endonucleases) – –modification specificity (methylation-sensitice restrictases) 6 7 RESTRICTION ENZYMES Sequence-specific DNA binding + cleavage Type I RE: 3 different subunits (specificity, methylation, nuclease) cleave outside binding sequence Type II RE: 2 identical subunits, both share spocific binding + nuclease cleave within the binding sequence (4-6 bp, palindromes) complementary methylases Type III RE: 2 different subunits, „something between“ 8 RESTRICTION ENZYMES Nomenclature: E Escherichia Genus co coli Species R RY13 Strain I první Order of discovery EcoRI 9 • sticky end formation EcoRI (GAATTC) – 5´-overhangs 5´ 5´ 5´ 5´ 5´ 5´ 5´ 5´ 3´ 3´ 3´ 3´ 3´ 3´ 3´ 3´ PstI (CTGCAG) – 3‘-overhangs NNNNNCTGCAGNNNNNNN NNNNNGACGTCNNNNNNN NNNNNCTGCAGNNNNNNN NNNNNGACGTCNNNNNNN NNNNNCTGCA pGNNNNNNN NNNNNGp ACGTCNNNNNNN 5´ 5´ 5´ 5´ 5´ 5´ 5´ 5´ 3´ 3´ 3´ 3´ 3´ 3´ 3´ 3´ RESTRICTION ENZYMES 10 • blunt end formation HaeIII (GGCC): NNNNNGGCCNNNNNNN NNNNNCCGGNNNNNNN NNNNNGGCCNNNNNNN NNNNNCCGGNNNNNNN NNNNNGG CCNNNNNNN NNNNNCC GGNNNNNNN RESTRICTION ENZYMES Isoschisomers: -recognize+cleave the same sequence X not necessarily in the same way -may differ in sensitivity to methylation (Msp I x Hpa II, Bstn I x EcoR II) Linear difussion mechanism: -efficient searching for binding sites by sliding along DNA; balanced non-specifc binding Star activity: -less specific cleavage under improper conditions (reduced water activity) 11 Methylation systems in v E. coli: • dam - 6N methylation of adenine GATC roles in mismatch repair, DNA replication, gene expression BamHI – GGATCC + (Bacillus amyloli) BclI – TGATCA - (Bacillus caldolyticus) MboI – GATC - Sau3AI – GATC + • dcm – inner cytosine methylation (5C) in CCAGG, CCTGG EcoRII - BstNI + • EcoKI – 6N methylation of adenine AAC(N)6GTGC, GCAC(N)6GTT • • For example, plasmid DNA cloned in E. coli dam+ is resistant to MboI METHYLATION-RESTRICTION SYSTEMS Bacterial defence against viral (phage) infection: methylated DNA is not cleaved by the restrictases, unmodified phage DNA is Post-replication maintenance DNA methylation in symmetrical sequences ……. mCG…. mCNG….. mCG…. ……. mCG…. mCNG….. mCG……… ..........GmC… GNmC….GmC… ….......GC…...GNC……GC…….. -bacterial DNA: hemimethylated after replication, methylation follows -phage DNA: non-methylated, cleaved -ca 0.2 % frequency of de-novo methylation: phage „learns“ how to survive and replicate 13 METHYLATION-SENSITIVE RE CG: HpaII mCmCGG (either of the mCs) CfoI GmCGC SmaI CCmCGGG TaiI AmCGT ClaI ATmCGAT CNG: MspI mCCGG CHH: Sau96I GG(A/T)mCmC 14 DNA POLYMERASES http://38.media.tumblr.com/c99435b8a277a5b815101853bf7a9d3d/tumblr_inline_n8t2khifnq1qg4nwx.png Polymerization ALWAYS 5‘→3‘! E. coli DNA polymerase I: 5‘→3´ polymerase 5´→3´ exonuclease (degradation ahead) 3´→5´ exonuclease (proofreading) http://img.sparknotes.com/figures/A/a0d1b3a1aaed46e29034f996722dd1a1/exonuclease.gif DNA POLYMERASES Proofreading exonuclease activity (removal of misincorporated bases) -labeling of 3‘-ends http://images.slideplayer.com/2/684881/slides/slide_32.jpg DNA POLYMERASES 5‘ → 3 ‘exonuclease activity 17 Klenow fragment: 5 ´ 3´ polymerase 3´ 5´ exonuklease NO 5‘→ 3‘ exonuclease!! (small fragment) „random priming“ DNA labeling http://www.thermoscientificbio.com/molecular-labeling -and-detection/biotin-decalabel-dna-labeling-kit/ DNA POLYMERASES 18 Terminal (deoxy)nucleotidyl transferase http://bioweb.wku.edu/courses/biol350/RestrictionEnz3/Review.html - statistical RNA/DNA polymarization without template - transfers nucleotides from dNTP to free 3´-OH end - selectivity control by metal ions: purine – Mg2+ pyrimidine – Co2+ - 3‘-OH overhangs (or ss NA) are prefered - labeling of 3´-ends 19 T4 polynucleotidyl kinase: - transfer of g-phosphate from ATP to 5‘-OH end of DNA/RNA - 5‘-end labelling Alkaline phosphatase (AP): 5‘-phosphate removal (restrictases and other DNases produce 5‘-phosphate) - prevention of vector/insert self-ligation - bacterial alkaline phosphatase (BAP) - calf intestinal phosphatase (CIF) C:\Documents and Settings\Biofyzikalni ustav\Plocha\ATP.png a b g 20 •5‘-labeling: polynucleotidyl kinase + ATP (g-phosphate is used) •32P, 33P, 35S (as thiophosphate); thiol-reactive labels • • • • • • • • •3‘-end labeling: DNA-polymerases or terminal transferase(TnT, TdT) (nucleotide including a-phosphate is used) •any labels attached to sugar or base •nick translation, random priming, PCR C:\Documents and Settings\Biofyzikalni ustav\Plocha\ATP.png a b g SUMMARY REMARKS TO DNA LABELING 21 Thermostable DNA polymerases: Taq DNA polymerase (Thermus aquaticus) temperature optimum 72 – 80 °C DyNAzyme, Vent, KOD... exo+/-.... fidelity versus speed versus tolerance to modifications https://upload.wikimedia.org/wikipedia/commons/thumb/9/96/Polymerase_chain_reaction.svg/2000px-Poly merase_chain_reaction.svg.png DNA POLYMERASES 22 Reverse transcriptase RNA-dependent DNA polymerase MuLV (Moloney murine leukemia virus) AMV (avian myeloblastosis virus) „SuperScript“: higher temperatures no RNaseH activity (RNaseH: exonucleolytic degradation of RNA strand in DNA/RNA hybrid) RT-(q)PCR analysis of gene expression 37 – 42 °C 23 DNA ligases Bacteriophage T4 DNA ligase: joining of sticky as well as blunt end cofactor = ATP E. coli DNA ligase: joins sticky ends bacterial ligases usually use NAD as the cofactor http://ocw.mit.edu/courses/biological-engineering/20-109-laboratory -fundamentals-in-biological-engineering-fall-2007/labs/mod1_3/ C:\Documents and Settings\Biofyzikalni ustav\Plocha\ligase.gif 1. step: transfer of APM to 5‘-phospho DNA end (the intermediate involves a diphosphate macroergic bond) 24 SINGLE STRAND SELECTIVE NUCLEASES In general: cleavage of both DNA and RNA Nuclease S1: endo- and exonuclease acidic pH optimum (4.5), Zn2+ ions -removal of ss overhangs, opening of hairpin loops -open local structures Mung Bean Nuclease: -similar to S1, milder conditions (less acidic) Nuclease P1: -neutral pH optimum, Zn2+ ions -ss-selective endonuclease + 3‘phosphatase -32P postlabeling analysis of DNA adducts Micrococal nuclease (MNase) -selective cleavage of untwisted DNA; AT-rich -chromatin digestion to (oligo)nucleosomes http://www.nwfsc.noaa.gov/publications/scipubs/techmemos/tm14/images/fig1.gif 25 SINGLE STRAND SELECTIVE NUCLEASES In general: cleavage of both DNA and RNA Nuclease S1: endo- and exonuclease acidic pH optimum (4.5), Zn2+ ions -removal of ss overhangs, opening of hairpin loops -open local structures Mung Bean Nuclease: -similar to S1, milder conditions (less acidic) Nuclease P1: -neutral pH optimum, Zn2+ ions -ss-selective endonuclease + 3‘phosphatase -32P postlabeling analysis of DNA adducts Micrococal nuclease (MNase) -selective cleavage of untwisted DNA; AT-rich -chromatin digestion to (oligo)nucleosomes http://mmbr.asm.org/content/75/2/301/F4.large.jpg http://www.biomedcentral.com/content/figures/1471-2199-7-37-5-l.jpg Single-strand selective enzymes •only detection of a open structure, not identification at the sequence level •often sufficient: evidence of formation of a expected structure •nucleases S1, P1, mung bean... cleave ss DNA (or RNA) •scDNA cleaved by S1, then restriction cleavage to map S1 celavage site S1 S1 restriction site restrictase restrictase http://www.nature.com/onc/journal/v23/n12/images/1207324f4.jpg agarose elfo distinct bands indicate site-specific cleavage by S1 Combination of chemical probes with S1 nuclease •chemical probes work within wider range of conditions than enzymes •modification of scDNA •then restrictase cleavage •chemical modification of bases in structure that existed in scDNA prevent formation of B-DNA •then S1 cleavage in the modified site S1 chemical modification restriction site restrictase S1 http://www.nature.com/onc/journal/v23/n12/images/1207324f4.jpg agarose elfo distinct bands indicate site-specific modification 28 NUCLEASES PROCESSING DNA ENDS Bal31 nuclease: cleaves both 3´ and 5´ ends in dsDNA -removal of ss ovehangs, cleavage in nicks and gaps -shortens blunt-ended dsDNA -identification of terminal DNA sequences Exonuclease III: degrades one strand in dsDNA (RNA strand in RN/DNA hybrid) from its 3‘-terminus -creates 5‘-overhangs Lambda exonuclease: degrades one strand in dsDNA from its 5‘-terminus -creates 3‘-overhangs http://cfile6.uf.tistory.com/image/2546B035552B5D86246763 (c) Lambda-exonuclease 29 PHOSPHODIESTERASES DNA/RNA exonucleases Phosphodiesterase I from snake venom cleaves 3‘→5‘ requires 3‘-OH produces 5‘-dNMP Phosphodiesterase II from spleen cleaves 5‘→3‘ requires 5‘-OH produces 3‘-dNMP 30 RNase A: endoribonuclease cleaves preferentially ssRNA „after“ Py, produce 3‘-phospho ends extremely stable, no cofactors, difficult to inactivate DNA purification (RNA removal) SNP mapping in DNA (RNase protection assay) http://www.gene-quantification.de/mrna-fig-2.gif http://www.gene-quantification.de/mrna.html 31 DNase I: DNA endonuclease cleavage of dsDNA or ssDNA to ~tetranucleotides requires Mg2+ (inhibition by EDTA), neutral pH optimum reductive inactivation (-S-S- bond stabilize structure), Ca2+ protection in dsDNA creates single-strand breaks, 5‘-phospho ends RNA purification (RNase-free!!), protein-DNA footprinting DNase II: double strand breaks, 3‘-phospho ends, no cofactors, acidic pH optimum DNase I Footprinting Analysis 32 chemical energy preserved Polynucleotide phosphorylase -random polymerization of ribonucleotides from NDPs -RNA degradation by inorganic phosphate (not hydrolysis) into NDP -reversible -polyribonucleotide biosynthesis -used during genetic code solution Phosphorylase does not phosphorylate! Engineered nucleases for genome editing 34 http://aws.labome.com/figure/te-164-2.png http://www.labome.com/method/Genomic-Engineering.html ZNF, TALEN -constructs of a nuclease (fok I) with DNA binding domains (addressing cleavage to a specific sequence) -analogous construct with DNA methylases, histone (de)acetylase: epigenetic modifications 35 Genomic Engineering Figure 3 http://www.labome.com/method/Genomic-Engineering.html CRISPP clustered regularly interspaced short palindromic repeats RNA-directed DNA celavage 36 http://download.cell.com/images/journalimages/0167-7799/PIIS0167779913000875.gr2.lrg.jpg Modifications of genomes Gaj et al. Trends Biotechnol 2013 Repair of the (ZNF, TALEN, CRISPP-induced) double strand breaks via NHJ of HR: -insertions, deletions – gene inactivation -inversions -transgene insertions, modifications of genes (precise by HR) 37 http://1.bp.blogspot.com/-5OsCXQgsOS8/Tje3gxHodQI/AAAAAAAAAHU/qwzkPrcYI6I/s640/Cloning_2.gif http://worldofbiochemistry.blogspot.cz/2011/08/cartoon-about-cloning-2.html