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Week 1 modules Module 1: DNA Structure What is the hereditary material? ● Chromosomes are composed of proteins and DNA ○ Proteins are comprised of 20 amino acids ○ DNA are composed of nucleotide bases, building blocks ■ It's a double helix ■ Bound together by complementary base pairs Griffith- 1928 ● British researchers investigating a strain of diplococcus bacteria that cause pneumonia in mice and in humans ● He had 2 different strains of the bacteria ○ The rough nonvirulent R strains ○ The smooth virulent S strain ● The rough nonvirulent type, the mouse would not develop pneumonia, the smooth virulent type the mouse would develop pneumonia and die ● When we grew the bacteria in culture he named them after their appearance ○ Extra purple coat in smooth was a sugar coat that was lacking in the rough strain ● Takes the type S bacteria and heats them up and kill the bacteria, then he injected mice with the heat killed bacteria, so we see no disease ● Then he takes the type S heated dead bacteria and combines them with the living type R bacteria, the mice develop pneumonia and die ○ When he examines the blood, he sees living type S bacteria ● Proposed the principle of transformation : genetic information from heat-killed S strain cells were transferred to the R cells, transforming them to virulence ○ The pollyschride coat allows the bacterial strain to hide from the host immune system, without it the mouse and human immune system would recognize the bacteria as a foregn invader and eminitae it ○ The information that was transfered was how to make that coat Avery, MacLeod and McCarty- 1944 ● They isolate components of heat-killed S cells and add purified molecules to living R type cells ○ Adding only protein or nucleic acid, etc. ○ Do these different components have the capability to transform these type R cells into S, giving them the ability to make the sugar coat ○ Carbohydrates/polysaccharides- NO ○ Lipids- NO ○ Proteins- NO ○ RNA- NO ○ DNA- YES! ■ DNA contains the information that the cell was using in order to instruct it how to make the polysaccharide coat ■ Added DNAse (cuts up DNA) to see if it ruins the cell information to form the coat, and yes it did ruin its ability, so only intact DNA can do it
Hersey and Chase- 1952 ● DNA is the hereditary material ● Use a bacteriophage to investigate DNA further ○ Composed of a protein coat and nucleic acid core ○ Protein “head” surrounds nucleic acid (DNA) ○ Can label the protein and DNA associated with the bacteriophage ● Growing bacteria in the presence of phage (viral) particles and radioactive sulfur (labeling protein) and in the other one with radioactive phosphorus (labeling DNA) ○ Put the bacteria in a fresh flask with bacteria not grown with the labels ● They found that phosphorus (DNA), but not sulfur (protein) entered the bacterial cell from the labeled bacteriophage ○ Captured viral particles that were generated after infection, broke them open and found that DNA was present and not sulfur, so DNA was passed to the next generation- hereditary material Hereditary material must be able to: ● Has to be chemically stable ○ Remain stable and be passed to daughter cells and future generations ● Has the ability to store a great amount of information (code for all traits) ● Have the capacity for change ○ Mutation to occur to generate new characteristics to have evolution Discovering the structure of DNA: ● DNA is a nucleic acid ○ A single building block is called a nucleotide ○ Nucleotides are composed of a sugar (deoxyribose), phosphate group (phosphorus atom bonded to 4 oxygen atoms) and nitrogenous base ○ 4 different nitrogenous bases: adenine, guanine, cytosine and thymine ○ 2 different types of sugar (deoxyribose- DNA) and (ribose- RNA) ■ Ribose has an OH group on second carbon, deoxyribose has a H group on second carbon ■ Count the carbons clockwise (5-membered ring, 5 carbons) ● 5th carbon covalently bound to 4th carbon ■ 1 prime carbon is attachment for nitrogenous base, 2 prime identifies the sugar, 3 prime has an OH group that allows individual nucleotides to join in nucleotide chain, 5 prime carbon is attachment point of phosphate group ● Nitrogenous bases ○ Purine (2 ring)- adenine and guanine (“ P ure a s g old”) ○ Pyrimidines (single ring)- cytosine, thymine and uracil (only RNA) Erwin Chargaff- 1984 ● Involved in investigating the proportion of purine and pyrimidines ● He always saw a 1:1 ratio of purine and pyrimidine bases ● Identified the amount of adenine was equal with the amount of thymine in a organism and the amount of guanine was equal with the amount of cytosine
■ Stabilizes the strands ● Strand synthesis ○ Primase ■ Gets the store ready for DNA polymerase ■ Type of RNA polymerase only used in DNA replication ● Can start synthesis of new nucleotide chains from scratch, a short RNA stretch 10-12 nucleotides long ○ DNA polymerase ■ Responsible for actually putting together individual nucleotide building blocks ■ It can initiate from scratch the synthesis of a new chain, needs primase to help it get started ■ Needs to have an exposed unpaired base and a 3’ hydroxyl group in order to start catalyzes covalent phosphodiester bonds between nucleotides on a new chain ○ Ligase ■ Joins Okazaki fragments and seals gaps in the sugar-phosphate backbone Leading vs. lagging strand synthesis ● Leading strand ○ The 3’ end OH group is exposed in the direction of the unwinding DNA, you can just keep on adding ○ Its continuous ● Lagging strand ○ Has to be constructed in fragments and eventually they are joined together ○ When DNA polymerase reaches the RNA primer it cleaves it and removes it puts down correct DNA nucleotides ○ Happens slower ○ Fragments are called Okazaki fragments PCR ● The amplification of a particular DNA fragment, a particular locus within the genome ● Allows us to separate out a particular locus in the genome from other loci ● Over 3 billion base pairs in the human genome ● Using DNA primers for PCR ● Taq polymerase ○ Special DNA polymerase used in PCR ○ Withstands the heat in denaturation ● Ingredients in PCR reaction (in vitro-lab) ○ Template DNA- extract DNA from organism of interest ○ DNA polymerase- heat stable taq polymerase ○ dNTPs- the nucleotides to make the new strand ■ Cleave two of the three phosphate to create energy to create new strand, phosphodiester bond ○ Buffer- stilts to have the right pH for taq polymerase to function properly ○ Primers- form complementary base pairs with target sequence on template strand ■ Hybridized with the template
■ Annealed to the tempkate ● Put all the ingredients in a thermocycler, 30-40 cycles of 3 different steps ○ Start with a double stranded DNA template, original DNA in original conformation ○ Step 1: denaturation ~94 ℃ ■ Heat up the reaction mixture and break apart the hydrogen bonds ■ Now have single stranded nucleotide chains ○ Step 2: annealing ~55 ℃ ■ Quickly cool the reaction mixture ■ Short primers find complementary binding region ■ Primer is the beginning of the new chain being made, so it starts off and remains in the strat it beings in, can be used to synthesize any other new strand, it gets in coorportated ■ Reform hydrogen bonds ○ Step 3: extension ~72 ℃ ■ Need to warm it back up a little for taq polymerase to bind and elongate the primers ■ Elongating the strand, making a copy (5’ to 3’) ○ In the end can end up with a billion copies after all the cycles Breast Cancer ● 5-10% have hereditary form of the disease, a mutation in BRCA1 gene or BRCA 2 ○ Inherent 1 nonfunctional copy and 1 functional copy are at an increased risk ● About 12% of women in the general population will develop breast cancer ● 55-65% women with BRCA1 or BRCA2 mutation will develop breast cancer (increased risk) ● Ovarian: 1.5% without mutation to ~39% with BRCA1 mutation (inherited mutation) ● How can we predict susceptibility? ○ Need to see what type of mutation is there can what does it change ○ Need a way to isolate only he BRCA1 gene out of the 3 billion base pairs ■ PCR and primers- isolation and amplification of a gene of interest DNA sequencing: Determine the identity/order of bases in a DNA molecule ● Start with the PCR product (amplicon) ● Ingredients in a sequencing reaction ○ Template DNA (PCR amplicon) ○ Sequencing primer ■ Only 1 of the two PCR primers, doesn't matter which one ○ 2 types of nucleotides ■ dNTPs- regular base pairs ■ ddNTPs- add these because fluorescently labeled, allows to ability to automate the process ● They remove the oxygen in the 3’ OH and terminate the chain, can’t form phosphodiester bond ● Small amount ○ Buffer ○ Taq DNA polymerase
○ Decreases cost, time, complexity Challenges of DNA replication specific for eukaryotes ● DNA is packaged in nucleosomes ○ DNA wrapped around a core of 8 histone proteins ○ Nucleosomes allow for the packing of DNA until we get the highly conserved form of the chromosome ○ During the process of replication need to be able to disassemble the nucleosomes ● Chromosomes are linear ○ A gap is left at the tips when RNA primer is removed ○ Maintaining the length of the telomere is hard ■ Loose length because of RNA primers when they get cut out ■ Telomerase is responsible for maintaining the length of our chromosomes Humans are diploid (2N) ● 23 chromosome pairs ● 22 pairs of autosomes ● 1 pair of sex chromosmes ● Homologous chromosomes ○ Contain the same genes, but they can have different version of those genes in the form of different alleles Telomerase ● Protein RNA complex ● Short stretch of RNA that has the ability to pair with the telomere sequence ● We can elongate/ extend the telomeres ● Composed of protein and RNA!!! ● Activity ○ Proliferating cells ○ Mutations that can affect telomerase function that can cause premature aging diseases ○ Telomerase is expressed in 90% of cancers Module 3- mRNA Transcription, Processing and Translation Gene expression ● Decode information in genes to produce molecules that determine phenotypic traits of organisms ○ Structures that determine phenotypic traits of organisms ● Process ○ In the nucleus: transcription ■ Copy of the gene or information in the gene ■ RNA transcript, DNA cannot exit nucleus ■ Processing/ splicing ● Coding and noncoding regions in the RNA
● Noncoding serene needs to be spliced out, then join coding segments back together ○ Take the transcript and exit the nucleus into the cytoplasm of the cell where you can translate the info ■ Translation : generate a corresponding polypeptide chain of amino acids covalently joined together ■ Polypeptide chain: allow for different folding of that chain into a protein ■ Fully folded protein have different structures, primary, secondary, tertiary, quaternary Transcription ● The events associated with the synthesis of an RNA molecule front eh DNA that constitutes the gene ● Transcription unit is the gene, the DNA ● RNA synthesis ○ Need to have a ssDNA template ○ rNTPs ○ RNA polymerase- enzyme responsible for contracting the mRNA transcript ■ We use RNA polymerase 2 ● pre-mRNA processing ○ Once RNA polymerase constructs molecule prior to leaving the nucleus ○ Needs to go through intron.exon splicing ■ Exon- coding portion, containing information to construct polypeptide/ protein ■ Introns- non-coding regions, spliced out ○ 5’ cap ○ 3’ polyA tail Differences between DNA and RNA: DNA RNA
- Double stranded - Single stranded
- Sugar: deoxyribose (2’ H) - Sugar: ribose (2’ OH)
- Bases used: A,C,G, Thymine - Bases used: A, C, G, Uracil
- Stores RNA, protein encoding info, transferred info to daughter cells - Carries protein-encoding info and help to make proteins mRNA ● Carries information that specifies a particular protein ● Three mRNA bases in a row form a codon, which specifies a particular amino acid ● Most mRNAs are 500-4500 bases long ● Differentiated cells produce certain mRNA molecules called transcripts ○ Information in these transcripts is used to manufacture encoded proteins ○ Contains the words that tell what the amino acid order in the polypeptide chain
● Initiation ○ TATAAT sequence, TBP will bind and initiate the unwinding of DNA and transcription factors bind and recruit/position RNA pol 2 ● Elongation ○ transcription bubble forms, RNA pol 2 constructs primary mRNA transcript, travels downstream ○ Creates pre-mRNA, before it's processed ● Termination ○ mRNA transcript released and RNA pol 2 dissociates from DNA Simultaneous transcription of mRNAs ● Several mRNAs can be transcribed all at once from the same template DNA strand at the same time ● Was can amplify the message, especially if the cell needs multiple copies of the same protein Concept of a gene ● Not all DNA is contained in a transcription unit is going to contribute the the mRNA molecule ○ EX: promoter sequence, enhancer sequences ● Gene is defined as the total DNA sequence required to encode an mRNA molecule ○ Incides coding portion and non-coding regions ○ Coding- exons spliced together ○ Non Coding- introns spliced out, promoter and enhancer sequences Genes in pieces ● Join together coding portions of the transcript ● Exons- DNA that encodes polypeptide (capital letters) ● Introns- DNA intervening between exons that does not encode polypeptides (lower case letters) Splicing ● Primary RNA transcript, pre-mRNA sequence ● Removal of INTRONS in the nucleus ● Sequence at exon-intron boundary specify locations for splicing ● Mature of cytoplasmic RNA exit the nucleus Why intron splicing? ● Eukaryotic specific character ● Can modify transcripts- building blocks ● Genome complexity Alternative splicing ● Selective combining of exons ● Increases complexity ● Many outcomes from 1 gene ■ Can splice exam 1,2 and 4 vs, 1,2 and 3 ■ Form different folding of proteins, different protein isoforms with different roles mRNA modifications ● Transcription in the nucleus ● Translation takes place in the cytoplasm ● Before the mRNA leaves the nucleus it is modified
○ Removal of introns ○ 5’ end methyl cap ■ Essential for initiation of translation ■ Small ribosomal subunit bind to recognize RNA transcript at this position and travel downstream until it encounters the start codon for synthesis for translation to occur ■ Protection of the mRNA transcript ■ Added immediately during transcription by an enzyme coupled to RNA pol 2 ■ Protect from greagation by RAT ■ Not added to non coding RNA (rRNA, rRNA) ○ 3’ PolyA tail ■ Adedines that do not correspond to complementary thymine ■ Added after the transcript has been generated ■ Longer the polyA tail, the more stable the molecule is and will remain the cytoplasm of the cell and translated to protein Translation ● The synthesis of every protein molecule in a cell is directed by an mRNA originally copied from DNA ● Information-transfer process: ○ Contained within the DNA, transcribed into mRNA that leaves the nucleus into the cytoplasm and then translated into protein ● Chemical process in translation where we are linking amino acids together into covalently attached polypeptide chain Translation: building a protein ● 3 bases specify 1 amino acid ● Linear sequence used in the form a triplet codons and specifies for specific structure/ folding of the protein 3 kinds of RNA ● Processed mRNA transcript ○ RNA pol 2 ● Ribosomes (delivered from rRNA) ○ RNA pol 1- NOT translated ● Transfer RNA (tRNA) ○ RNA pol 3- NOT translated mRNA ● Rapid turnover ○ They get degraded and created fast ● Heterogenous size ○ Different sizes and different messages for different proteins ● Different cells have different populations, different proteins to do their jobs Ribosomes ● rRNA is the same in all of the cell types
○ In weeks after fertilization there is an increase in the alpha globin gene and once it gets tired and and expressed it really maintains its expression throughout the lifetime of an individual, but after fertilization there is production of the gamma chain and that starts to decrease when the beta globin chain starts to be produced ○ Why do we need different hemoglobin molecules? ■ Affinity for O2, fetal structure has a higher affinity for oxygen than the adult and that is important because the fetus relies on the O2 from maternal blood pools in the placenta, if not the fetus could be deprived Sickle cell anemia ● Mutation in the beta globin gene- adult hemoglobin molecule ● If an individual inherits two copies of the non-functional beta globin gene then their hemoglobin molecules are pre to clump together and cause the red blood cell to clog the capillaries and degradation causing anemia ● activating the transcription or silencing it ● Altering gene accessibility through chromatin modifications ○ Chromatin is composed of DNA and protein (histone) ○ Condensing or relaxing the chromatin Vitamin D Response Element (VDRE) ● Particular conserved sequence we find in the promoters of genes (like TATAAT sequence) ● Some genes have a VDRE ○ Start with an external signal and transcription factors connect the genome expression of genes with external signals ○ Vitamin D binds to the VDR, vitamin D will carry that message into the nucleus and activate transcription if target genes, genes that are responsive to the presence of vitamin D, activate expression of Vitamin D regulatory genes ○ Activate transcription of those particular genes when the appropriate eternal cellular message has been received DNA is packaged into nucleosomes forming chromatin ● Whether or not the sequence is actually acceptable to access ● Histone proteins and DNA forming chromatin ● DNA is wrapped around a core of 8 histone molecules= nucleosomes and can all be packed in the form of a chromosome ● Chromatin is a problems for gene expression, but provides opportunity to regulate gene expression ○ Chromatin remodeling ■ Various protein complexes called remodeling complexes responsible for repositioning the nucleosomes to provide access to a particular DNA sequence ■ Moving or repositioning of a nucleosome along the length of DNA to provide access or restrict a regulatory factor ○ Histone modification ■ N terminal tails sick out and are necessary for regulation ■ post -translational modifications
● Add different chemical groups to amino acids in the polypeptide chain, modify hoe accessible a particular gene is for transcription ● Can add acetylation, methylation and phosphorylation to N terminus ○ Acetylation : lysine is typically one of the positions that has the capacity for being modifies, acetyl groups or take them away and will alter accessibility of associated genes for transcription ○ Lysine carries an overall + charge, while DNA carries an overall
- charge, a lot of lysines in N terminal tails (N terminal tails are + and opposites attract) and they bind DNA tightly if not acetyl group on them, but if you add acetyl group, it will shield the + charge and allow the N terminal tails to release the DNA, not as tightly bound and allows the chromatin remodeling complex to work ○ Enzymes involved: HATs (add acetyl groups to N terminal lysine aa) and HDACs (remove the acetyl group) ○ DNA methylation- epigenetic programming ■ 5’-CG-3’ ■ Wherever there is a C upsteam G, it can be methylated ■ CpG dinucleotides (p=phosphodiester bond) ■ Promoter regions contain high numbers of these bonds so they are called CpG islands ■ Give us an opportunity to turn on and off genes ■ Addition of a methyl group by methylase or DNA methyltransferase (DNMT), bound to the C 5’ to a G ■ Open lollipop= no methylation (expression), closed lollipop= methylated (silencing) ■ How does this work ● Start off with a DNA molecule that is unmethylated ● De-novo DNA methylation- sequence that was not previously methylated and DNMTs that bring on the methylation ● Top strand is 5’-3’, bottom strand complementary and antiparallel ● CpG on top also on the bottom, 2 methyl groups bound, one on each stand ● Methylation occurs during early development, then it is maintained all the daughter cells will also carry that methylation ● When you silence a gene something called and MBP will recognize the methylation and then receipts HDACs (remove acetyl group) and will close the chromatin, turning it off or silencing gene expression Summary: Transcription ON Transcription OFF
- Active chromatin - Condensed or inactive chromatin
○ Prader willi syndrome ■ Hypotonia at birth and delayed development, poor muscle tone, excessive appetites later in childhood and can lead to obesity, ■ Chromosome 15 parental deletion ○ Anglelman syndrome ■ Nervous system function layered, delayed development, etc. ■ Chromosome 15 maternal deletion Genomic imprinting ● Sex specific improting patters that we pass to offspring ● igf2 - human chromosome 11 ○ Paternal allele is passed in an active state and the maternal allele is epigenetically silenced ○ Only 1 copy of the gene can be expressed ○ Important for determining size of placenta ● Male pass male imprinting patterns and females pass female imprinting patterns Therapy ● Can we turn off paternal/ maternal copies? cDNA Microarrays ● Simple way to examine levels of gene expression ● Have a plate where we have fixed single stranded probes attached to the plate that are complementary to a particular gene ● Harvest the RNA from each of the cells and quickly convert it to DNA because RNA is unstable ○ Call it cDNA, more stable ○ Different than the DNA because the introns have been spliced out ○ Also label it was the fluorophore ○ Combine them all together and expose them to the plate ■ All these different spots on the plate ■ RED is mRNA expressed in the cancer cells ■ GREED is mRNA expressed in the normal cell ■ YELLOW is the gene is expressed in both the normal and cancer cells ■ No color means the gene wasn't turned on at all Gene expression can be controlled at many levels ● Chromatin structure ● Transcription ● Alternate splicing ● mRNA stability ● MicroRNAs (block protein synthesis) ○ Short 20 bases in length ○ Post Transcriptional modification ○ Eliminate mRNA transcript ○ Complementary base pair to mRNA ● Posttranslational modifications
○ Additions to the protein that will modify is its in an active state or targeted for degradation RNAi ● Similar process to microRNAs, but this one is utilized in a lab for research purposes ● Introduce a double stranded RNA molecule in a cell, short and the cell DOES NOT like it, thinks its a virus ● Enzyme dicer recognizes it and cuts up the double stranded RNA into fragments ● The fragments bind to another protein called the RISK complex ○ Retains a single strand of the double stranded RNA and it travels around the cell reading mRNA ○ Wherever it find a complementary mRNA target it's going to cleave it ○ Has nuclease capabilities- cut a nucleotide chain ○ Corresponding mRNA is no longer expressed ● Gives us a was in in-cell culture to actively silence whatever gene we want to silence, do this by degrading corresponding mRNA complexes ○ Be able to study function because it silences the expression of a particular protein Pseudogenes ● Lost their function ● Non-functional, not translated ● Sequences very similar to known genes, but are not translated Transposons ● Move ● “Jumping gene” ● Most abundant repeat sequence of DNA ● 45% of genome ● Common in primates Viral DNA ● Present in our genome ● 8% ● HERVs ● Reverse transcriptase can make DNA from RNA Module 5: Mutation (SNPs and STRs) Mutation ● Some change or alteration in the sequence of a particular locus in your genome ● Ex: peppered moth ○ peppered morph vs. dark melanistic phenotype ○ Trees were covered heaving with lykin and allowed the peppered moth to survive because they can camouflage themselves to the tree ○ Industrial revolution caused lykin to die off an expose the dark bark and then the dark melanistic version increased in size because now the predators can easily see the peppered moth ○ Both color morphs already existed before industrial revolution
○ Alters the reading frame, affects everything downstream, SEVERE!! ○ Frameshift mutation ● Base deletion ○ Take one of the nucleotides away ○ Alterns the reading frame, affects everything downstream, SEVERE!! ○ Frameshift mutation Biological effects ● Nonsense mutation can be most deleterious or damaging for the protein to proper functionally and the corresponding phenotype ○ Location in the protein is important, the larger the portion lost the more damaging the mutation was ● Same sense or missense are not deleterious ○ Altering the amino acid for one specific location Sickle Cell Anemia ● Single point mutation that causes this phenotype ● Mutation in the beta globin gene ● G A G goes to G T G, glutamic acid vs. valine ○ Hydrophilic glutamic acid to hydrophobic valine ● Consequence ○ Mutation is exposed to cytoplasm ○ When you switch to hydrophobic causes the clumped hemoglobin and generates cycle form in red blood cells ○ Anemia, impair blood supply to various organs and spleen enlargement Mutation: effect on gene function ● Altered level of expression ○ Doesn't always have to be a mutation in the coding region of a gene, could be in the promote region ● Loss of function ○ The protein is not expressed at all, no longer can be transcribed ● Gain of function ○ Gene activation where there shouldn't be ○ The protein could be doing a job it's not supposed to ○ Cancer development Mutations can be ubiquitous or conditional ● Ubiquitous- always present ● Conditional- phenotypic change can only be observed under permissive conditions Transitions/ tranversions ● Spontaneous point mutation ○ Bias in favor of transitions vs. transversions ○ Ration approximately 2: ● Why? ○ Same number of rings ● Consequence
○ Maintain the distance of three rings across with regard to complementary base pairing ○ Not as easy to spot that change by DNA repair machinery and can be passed on to daughter DNA molecules What causes mutations? ● Spontaneous, random, unpredictable ○ Errors in DNA replication ○ Tautomeric shift ■ rare unstable form of the 4 bases that can occur when we have a change in the position of protons associated with that nitrogenous base ○ If the base in the unstable tautomer form during DNA replication, we have abnormal paring, wrong paring partner ○ Wobble base pairing : thymine can form base pairs with guanine and cytosine with adenine base to 3D nature of DNA molecule ■ Because of flexibility of structure allows for wobble pairing or misparing ■ Why it's been proposed as the cause of the mutation because we have seen this before in out cells- degeneracy of the genetic code ● Mutation will not always lead to amino acid change ● Induced ○ Chemical mutagen/ radiation ○ Base analogs ■ Chemicals that become incorporated into DNA chain that look like bases but will cause misparing instead ■ Chemical gets mistaken for nitrogenous base ○ Induced single base mutations ■ DNA modifiers that act directly on DNA, but don't directly get incorporated ■ physical - only radiation ● Ionizing radiation causes breaks in the covalent bonds in the DNA, can have double stranded breaks that can be deleterious ● UV light causes a thymine dimer, where 2 thymine molecules that are adjacent to each other are , they can be covalently bonded to one another and a complication in allowing for transcription ● Affects excision repair system ■ Chemical- ● alkylating agents, deamination, oxidative reactions ○ Alter the nitrogenous base, so they allow some misparing to occur ● intercalating agents ○ insertions/ deletion by inserting themselves in the rungs of the staircase, nitrogenous bases Many types of damage ● DNA polymerase can proofread and fix mistakes as strands are being synthesised
