Molecular Biology I: Nucleic Acid Metabolism Lecture 06 (2024 S1)

MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Lecture 06: Molecular biology techniques 1 Recap of last lecture: Eukaryotic DNA replication – initiation and elongation:...

MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Lecture 06: Molecular biology techniques 1 Recap of last lecture: Eukaryotic DNA replication – initiation and elongation: 1. Licensing 2. Activation 3. Firing 2 Recap of last lecture: Okazaki fragment maturation: In eukaryotes, RNA primer is removed that RNase H1 and FEN1 (5’ – 3’ exonuclease), and gap is filled by DNA pol d 3 Recap of last lecture: Comparison of prokaryotic vs. eukaryotic DNA replication: Prokaryotes (E. coli) Eukaryotes Occurs in cytoplasm Occurs in nucleus Single origin of replication Many origins of replication DnaG makes RNA primer Pola makes RNA and DNA primer Pol III = main replicative Pold synthesizes lagging polymerase strand; pole synthesizes leading strand RNA primer removed by RNA primer removed by RNase H RNase H1 and FEN1 Replicates naked DNA Replicates DNA in chromatin context Okazaki fragments are 1000 – Okazaki fragments are 100 – 2000 nts in length 200 nts in length Replicates at 750 – 1000 nts Replicates at 50 – 100 nts per per second second 4 Recap of last lecture: Replication fork of eukaryotes: Nucleosomes are recycled during replication – old nucleosomes (dark green in the figure below) are roughly evenly divided and recycled onto the two daughter duplexes. New nucleosomes (light green ones) are also assembled at gaps. 5 Recap of last lecture: Eukaryotic mismatch repair: General process is similar to prokaryotic mismatch repair; however, does not scan for methyl to determine which is the template strand. Instead, scan for nicks on newly synthesized strand and chew back from the nicks. 6 Recap of last lecture: End replication problem: Chromosomes of eukaryotic cells are linear – so the DNA replication machinery cannot replicate the very end of the lagging strand. Eukaryotic chromosomes end in direct repeat sequences called telomeres. In human, repeats = (TTAGGG)n 7 Recap of last lecture: Connection between telomere erosion/shortening and aging: Telomeres in somatic cells (e.g. leukocytes) shorten over an organism’s life span (measured by Terminal Restriction Fragment assay or FISH-quantification assay) Telomere hypothesis of cellular aging à telomere length = biological clock for aging? Note the difference between “chronological” vs. “replicative” aging 8 Recap of last lecture: Protection of telomeric ends: Exposure of telomeric ends can trigger DNA damage/repair checkpoints. Therefore, telomeric ends must be protected. Protection mediated by formation of T- and D-loops D-loop and also by a variety of proteins bound to telomeric sequences 9 Recap of last lecture: Chromosome-end fusions due to critically short telomeres : DNA damage- response/repair proteins 10  Recap of last lecture: Telomere shortening and reactivation of telomerase: Telomere erosion correlates with cellular (replicative) aging and increasing number of cell division/population doubling. AKA Hayflick’s limit or M1 AKA M2 11 Recap of last lecture: Tetrahymena as an enriched source of telomerase Tetrahymana = single cell protozoan (like paramecium). Macronucleus contains almost 10,000 individual linear mini-chromosomes, each with telomeres à require highly active telomerase to make all these telomeres. Perfect source for biochemical purification of telomerase. 12 Recap of last lecture: Telomerase activity: Greider and Blackburn first to identify telomerase activity in Tetrahymena extracts. Showed that activity has both RNA and protein components (activity is sensitive to RNase as well as protease digestion). RNA component = TER Protein component = TERT Note: human equivalents = hTERT and hTR Example telomerase assay gel result (NOT the original data from Greider and Blackburn) 13 Recap of last lecture: Telomerase activity: TER = long RNA that folds into complex secondary structure and binds TERT polymerase enzyme. Within TER, there is a region complementary to G-rich strand that is used as template for G-rich strand synthesis 14 Recap of last lecture: Telomerase activity: RNA template has sequence Telomerase is a reverse complementary to transcriptase that specifically G-rich repeat and extends the G-rich strand. telomerase adds 6 nts (one repeat) Telomerase activity made up of at a time protein component (TERT) = polymerase, and RNA component (TER) = template G-rich strand synthesis involves multiple rounds of DNA elongation and translocation of the RNA template/enzyme. C-rich strand synthesized by the regular cellular replication machinery using the extended G-rich strand as template. 15 Recap of last lecture: Telomerase function: Telomerase extends both leading and lagging strands Extends G-rich strand in both cases 16 Recap of last lecture: Immortalizing cells by hTERT expression: Expression of hTERT in somatic cells is sufficient immortalize those cells: Increases and maintains slightly longer telomere lengths Bypasses Senescence or Crisis 17 Recap of last lecture: Reagents important for cloning: 1. Enzymes: Phosphatases, kinases Nucleases à e.g. endonucleases, exonucleases, restriction endonucleases, RNases Restriction enzymes à useful for: Restriction enzyme mapping Cloning of DNA fragments à Need REs, vectors, antibiotics for selection, screening methods Ligase à join DNA fragments together 18 Recap of last lecture: Reagents important for cloning: Note what are compatible vs non-compatible ends for ligation: Note: EcoR1 and Mfe1 also have compatible ends 19 Recap of last lecture: Example of cloning (vector + insert): Sticky ends: Overhang DNA sequences must match (à need fully matching overhangs to be able to ligate together) Blunt ends: All blunt ends are compatible for ligation 20 Recap of last lecture: Example of cloning (selecting for plasmid-containing clones): Heat shock and transform into bacteria 21 Methods in Molecular Biology: Tricks of cloning: 1. Phosphatase treatment of vector: Useful for preventing re-ligation of vector vector CANNOT treat both vector and insert DNA fragment with phosphatase, otherwise WON”T ligate together 3’ 5’ insert 5’ 3’ 5’ 3’ 3’ 5’ 22 Methods in Molecular Biology: Tricks of cloning: 2. Blue-White screening: Multiple cloning site contains the lacZ gene which encodes the b-galactosidase gene. This enzyme can cleave X-gal to form intense blue precipitate. Successful cloning of insert disrupts the lacZ gene, resulting in loss of b-gal expression and, therefore, produce white colonies instead of blue ones. 23 Methods in Molecular Biology: Tricks of cloning (continued): Fill in overhangs to produce blunt ends: à since blunt ends will always be compatible for ligation with each other Can only fill in 5’ overhangs Need to use polymerase (e.g. T4 DNA polymerase) + nucleotides for fill in reactions 24 Methods in Molecular Biology: Tricks of cloning: Chew back overhangs to produce blunt ends: Usually when one wants to convert a 3’ overhand to blunt end Use enzyme such as Klenow fragment which has 3’ à 5’ exonuclease activity 25 Methods in Molecular Biology: Tricks of cloning: 4. Adding adaptors or linkers: For adding sticky ends to blunt ends. Can generate linkers by synthesizing short complementary oligos and anneal them together add ligase to ligate ends Linkers are also useful for adding known DNA sequences to DNA fragments with unknown sequences so that oligonucleotides complementary to linker DNA can be used as primers to sequence the unknown DNA fragment 26 Methods in Molecular Biology: How to test if a clone contains desired insert: 1. Harvest DNA and do restriction enzyme mapping. For example, after blue-white screening, pick white colonies and grow cultures from single colonies (get pure population all derived from single cell) Isolate plasmid DNA from bacterial cultures, cut with known REs that flank insert fragment or within insert sequence Run digested fragments on agarose gel to determine restriction map 27 Methods in Molecular Biology: How to test if a clone contains desired insert: 2. Harvest DNA and sequence DNA directly DNA sequencing gives the exact sequence of a DNA fragment For synthesis-based methods, need short oligo to act as primer for synthesis of new DNA that can then be detected or analyzed. Note that synthesis can only go in 5’ to 3’ direction By choosing primers to anneal to unique sequences at one or the other end of MCS (e.g T7 or Sp6 sequences in the figure), can sequence into inserted sequences to verify identity of insert DNA, and also to confirm no mutations were generated in the insert 28 Methods in Molecular Biology: DNA sequencing technology: Original sequencing method (Maxam Gilbert sequencing) uses chemicals to cleave ssDNA, and assemble DNA sequence based on the cleavage pattern from different chemicals that cut after specific nts Fred Sanger developed alternative method in 1977 called dideoxy sequencing, based on the use of dideoxy nucleotides (ddNTPs) that cause chain termination in in vitro DNA synthesis reaction How do these two nucleotides differ from one another? Still common method for sequencing to this day 29 Methods in Molecular Biology: DNA sequencing technology (Sanger sequencing): Also add 32P-labeled dNTP to label newly synthesized strand 30  Methods in Molecular Biology: DNA sequencing technology (Sanger sequencing): Perform 4 parallel reactions, each using one of the 4 ddNTPs Need high resolution gels to resolve each bp à Usually use 0.4mm thick polyacrylamide gels Direction of reading the sequence (5’ to 3’ of synthesized DNA strand) Typical sequencing gel autorad 31