Molecular Biology Notes WK4 PDF

DNA Mutation and Repair Dr Jack Sunter [email protected]  Learning Outcomes Explain the structure and organisation of DNA and the mechanisms of DNA replication and repair. Demonstrate an understanding of a range of modern molecular biology techniques....

DNA Mutation and Repair Dr Jack Sunter [email protected]  Learning Outcomes Explain the structure and organisation of DNA and the mechanisms of DNA replication and repair. Demonstrate an understanding of a range of modern molecular biology techniques. 2  Peppered moth Before the industrial revolution After the industrial revolution London smog and 1956 Clean Air Act  Types of Mutations Mutations change the sequence of DNA Point mutation ATTGCAA ATTACAA Insertion ATTGCAA ATTAGCAA Deletion ATTGCAA ATTCAA Transversion mutation: – purine (AG) is replaced by a pyrimidine (TC) – pyrimidine is replaced by a purine Transition mutation: – purine is replaced by a purine – pyrimidine is replaced by a pyrimidine  Mutation Mutations can occur in: Germ cells (sperm, eggs)  Can cause heritable defects Somatic cells (adult cells apart from germ cells)  Can cause diseases such as cancer Cells in the developing embryo  Can cause developmental defects  Mutation Mutations can occur in: Coding genes Can affect protein expression/structure Non-coding RNAs (eg. rRNAs, tRNAs) Can affect translation, transcription, splicing etc. Non transcribed regions (e.g. promoters, enhancers)  Can affect transcriptional Mutations can occur regulation  Effects on proteins 1. Nonsense mutation – truncates protein 2. Missense mutation – changes an amino acid 3. Frameshift mutation – changes No (silent) mutation Nonsense Missense Silent reading frame AAG TAG AGG ACG AAA 4. Lys Silent mutation STOP – does Arg not Thr alter the Lys amino acid Nucleophilic Basic Basic Conservativ Radical e replaceme substitution nt  Silent mutations No effect on amino acid sequence But can affect transcription or translation No mutation Silent More than one codon can code AAG AAA for a single amino acid Lys Lys Some organisms favour a particular codon for a given amino acid E.g. Of the four valine codons, Basic human genes use GTG four times more than GTA Known as codon bias Presence of a non-favoured codon (rare codon) can slow  DNA Mutations Mutations either: – Spontaneous – Induced  Spontaneous Mutations Mutations that occur during normal cellular processes Mutations are rare events and occur at a background level for any particular organism Mutations can be caused by: – Malfunction during DNA replication causes the wrong base to be inserted during DNA synthesis – DNA bases are modified  Replication errors Wrong bases inserted at DNA replication, or a DNA base is skipped over Errors are made by DNA polymerase about one mutation every 105 to 106 base pairs Corrected by proofreading activity of DNA polymerase Most replication errors are fixed by proofreading activity of polymerase Rate of incorporation slows dow using an 3’ - 5’ exonuclea 99% of mismatches fixed with Replication errors - DNA 5’ slippage 3’ During replication, the DNA polymerase and newly synthesized DNA strand complex sometimes temporarily dissociates from the template DNA In repetitive sequences, the polymerase may reassociate with the template strand in a position one or two repeats ahead or behind where it left off Lead to insertions or http://www.sciencedirect.com/science/article/pii/ DNA slippage Can lead to expansion of CAG in huntingtin gene  What type of spontaneous base modifications can occur? Discuss in pairs 10 – 15 minutes Report into google doc – type of modification? – what’s the consequence? 15 Antioxidants – protectors against free radicals Antioxidants donate an electron to free radicals, thereby reducing their reactivity  Damage in the DNA template can lead to double strand break (DSB) formation during replication ‘Nick’ = break in phosphodiester bond from oxidative damage Loss of DNA or translocatio ns  Double strand breaks can lead to chromosome translocations Chromosome translocations - piece of one chromosome become attached to another chromosome Some spontaneous translocations can lead to cancer progression  Induced Mutations DNA damage caused by exogenous agents - mutagens Discuss in pairs 10 – 15 minutes Report into google doc – type of damage/modification? – what’s the consequence?  Use of mutations in research EMS (ethylmethane sulfonate) and ENU (N-Ethyl-N- nitrosourea) are powerful mutagens (alkylating agents) Animal models can be treated with ENU Animals with a resulting phenotype can be isolated and the mutated gene identified by classical genetics Help identify genes involved with disease Paul Potter’s work involves this http://www.brc.riken.jp/lab/gsc/mouse/AboutUs/ Use of mutations in research e.g. ENU http://www.brc.riken.jp/lab/gsc/mouse/AboutUs/  (N-ethyl-N-nitrosourea) - alkylating ag Identify genes involved in disease EMS mutagenesis on Caenorhabditis elegans (cheap and quick to grow) Treat C. elegans worms to 0.05 M EMS at 20°C for 4 hours. Look at progeny: Wild-type small mutant (adults are small) Dumpy mutant (adult animals are short and thick) egg laying–defective mutant (adult animals rarely lay eggs, so embryos mature and hatch inside the adult) Identify genes involved in development – sequencing  Use of mutations in therapy Cancer cells divide more rapidly than most other cells DNA-damaging agents (radiotherapy, chemotherapy) have more effect in cancer cells than in most healthy cells Stop cancer cells from growing and spreading Chemotherapy – many types, e.g. mustard gas derivatives (alkylating agents) Side effects of chemotherapy or radiotherapy = induction of cancer http://cancerhelp.cancerresearchuk.org/about-cancer/what-is-cancer/cells/  DNA Repair DNA damage can occur from various sources, and if left unchecked will have disastrous consequences for an organism Fortunately, there are several DNA repair systems to maintain genetic stability There is far too much to cover in one lecture (we could do a whole Much of what module on we it!) know about – extra DNA repair reading… has been obtained through studying the  Repair of mismatches Mismatches arise during DNA synthesis Or caused by chemicals/oxidative damage 99% of spontaneous replication errors are repaired by proofreading by DNA polymerase this is the 3’ - 5’ exonuclease activity most of the rest are repaired by mismatch repair or excision repair  Mismatch repair Newly synthesised strand will commonly include mismatch errors even after proofreading activity Fixed by mismatch repair GATC G GATC In E. coli: T DNA is normally methylated at GATC sites on both strands Just after replication, only one strand is methylated MutS specifically recognizes and binds mismatch MutL assembles with MutS and activates MutH MutH binds nearest methylated GATC, differentiating between old strand and new strand Complex introduces a nick in the new strand Helicase displaces the nicked strand  Mismatch repair in humans Similar process but with more layers of regulation Eukaryotes have MutS and MutL genes (called MSH and MLH genes – first studied in yeast) MutS homologues MSH1, MSH2….MSH5 MutL homologues MLH1, MLH2 etc. Eukaryotes don’t have MutH Must have another enzyme for incision – still to be found  Mismatch repair and colon cancer hereditary non-polyposis colon cancer is a hereditary cancer (Lynch syndrome) 2% of all colon cancers early onset (40s) 35% have a defect in MSH2 65% have a defect in MLH1 Mutations that arise in this condition are small additions or deletions - resulting from ‘replication slippage’ Hairpins in newly synthesized DNA are detected by the mismatch repair system and removed  Excision Repair Involves excision of the damaged region and replacement through the use of the complementary DNA strand as template Two of the most common pathways for repairing damaged DNA: – Base excision repair – Nucleotide excision repair X = Damaged base Base excision repair Apurinic/apyrimidine (AP) E.g. Alkaylated bases (induced by endonuclease cuts phosphodiester alkylating agents), Deaminated bond bases (spontaneous or induced), Oxidized bases (induced by superoxide radicals) Deoxyribose removed by dRpase Repair Polymerase (such as PolI in E. DNA coli or Pol b in mammals) fills gap glycosylase and ligase seals DNA. removes base using undamaged strand as template http://www.funpecrp.com.br/gmr/year2003/vol1-2/ NB This is in E. coli  Nucleotide excision repair Repairs large changes in DNA double helix, such as ‘bulky lesions’ caused by intercalating agents or pyrimidine dimers Multienzyme complex (in bacteria UvrA, B and C) scans DNA for distortion Enzyme complex cleaves DNA backbone helicase on either side of distortion leaving a gap of about 12 nucleotides in bacteria (30 in humans) DNA helicase peels away a single- stranded oligonucleotide containing the lesion Large gap in the DNA is then repaired by DNA polymerase (DNA PolI in bacteria)  Xeroderma pigmentosum (XP) - human repair defect Severe sunburn after only a few minutes in the sun, freckling in sun exposed areas - caused by defects in nucleotide excision repair Problems repairing UV-induced damage ~50% of patients die of cancer before age of 20  DNA double-strand breaks DNA double-strand breaks are much more difficult to repair Sequence information may be lost Repaired by: Homologous recombination (HR) Non-homologous end-joining (NHEJ) Non-homologous end-joining In pairs identify the important proteins required for HNEJ 5 minutes and report back  Repair of double DNA is nibbled back by an exonuclease strand breaks by homologous Invasion of the recombination homologous sister Recombination repair chromatid Involves pairing damaged DNA with a homologous DNA The invading duplex, i.e. a template strand acts a Process is very complex primer for DNA Many proteins polymerase accumulate at the site ofNewly damage synthesised region anneals to the DNA on the DNA pol fills the gaps other side of the DSB Ligase seals the nicks Homologous Recombination Can we take advantage of the endogenous enzymes to recombine homologous molecules via a double stranded break in the target chromosomal sequence? Can we recombine in a desired sequence?  Basic idea of what we can use homologous recombination for… Homology arm Homology arm construct Genome before Genome after Can we introduce a double stranded break anywhere we want in the genome?  CRISPR/Cas9 system CRISPR – clustered regularly interspaced short palindromic repeat Bacterial defence system – ‘immunological memory’ Bacteria cuts foreign DNA (red/pink) and acquires it into CRISPR locus Bacteria transcribes the CRISPR locus to generate a guide RNA with a region, 20 nt, that is complementary to the foreign DNA (red/pink) Complementary region base pairs with the target DNA Cas9 endonuclease recognises this complex and cuts the DNA to introduce a double stranded break Wiedenheft et al. 2012 Nature 482:331-338 To cut the genome at a specific site CRISPR uses ~20 nt sequence of crRNA to bind its complementary sequence of exogenous DNA Additional trans-acting RNA (trancrRNA) recruits Cas9 endonuclease Cas9and crRNA cuts DNA trancrRNA can be synthesized into a single chimeric RNA - guide RNA (gRNA) https://www.thebestgene.com/CRISPRInfoPage  Genome editing using CRISPR/Cas9 Cas9 guide RNA microinjection into embryo Target genome e.g. Drosophila Create a knock out mutant Not just flies http://trialanderror-scienceblog.com/?tag=cas9  You can edit the genome of Drosophila, cell lines and cats….. What next? DNA mutations: the bad and good Fewer than 1 in 1000 accidental base changes in DNA results in a permanent mutation The bad: mutation in a proto-oncogene can lead to cancer The good: mutation confers advantageous traits – drives evolution Balance between evolution and disease