Chapter 12 - Gateway To Biology: Molecular Biology PDF

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Gateway To Biology: Molecular Biology
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Chapter 12 – DNA Structure, Replication and Organization  

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Characterizing DNA sequences can enhance our understanding of genetic history, but it is overshadowed by DNA damage and contamination The double helix of DNA is subject to o Breakages in one or both strands o Inappropriate cross-linking o Chemical modification of individual bases DNA can be ‘maintained’ via very cold temperatures, as ancient bacterial DNA sequences have been recovered from 500000 year old sections of ice cores DNA naturally degrades over time, so aDNA sequences remaining in a tissue sample are rare and prone to contamination, which is why handling DNA needs to be done in a very clean, contaminant free fashion Miescher collected pus cells from bandages and extracted an acidic substance with a high phosphorus content, and called it nuclein o Nuclein, 50 years later, was identified as DNA – the genetic material of all living or dead organisms Watson and Crick used an array of processes to assemble a molecular model of DNA that enabled scientists to understand key processes in cells in terms of the structure and interaction of molecules, such as storage of information and DNA replication 12.1 – Establishing DNA as the Heriditary Molecule

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Proteins were believed to be hereditary molecules because they has 20 types of amino acids vs nucleic acids’ 4 nitrogenous bases, so proteins seemed to offer greater opportunities for information coding Griffith experimented with two strains of the bacterium streptococcus pneumonia (causes pneumonia) o The S strain has a polysaccharide capsule around each cell and was highly infective when injected into mice o The R strain was avirulent and the mice lived, so the S capsule hindered the immune system o When the S strain was heated and killed and injected with the R strain, the mice died o So the R bacteria acquired the ability to make the deadly capsule and change to S strains, and the inherited by the descendents of the transformed bacteria

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o This is a transformation and the agent responsible is the transforming principle The nature of the molecules responsible for the transformation? o Couldn’t be a carb or lipid cause the structure is repetitive and not likely to carry information o Proteins and nucleic acids are built of unique combinations making them likely candidates as “information carriers” 12.2 – DNA Structure

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DNA contains four different nucleotides each consisting of: o A 5 carbon sugar o A PO4 group linked to the 5’ carbon on the sugar o A nitrogenous base linked to the 1’ carbon on the sugar  A and G are purines, a pair of fused rings of carbon and nitrogen  T and C and pyrimidines, a single carbon ring  Chargaff’s Rules  # of adenine = # of thymine, and same with G and C o Nucleotides join to form polynucleotide chains, where the deoxyribose sugars are linked by PO4 groups making the sugar-phosphate backbone, the PO4 groups are the bridges between the 3’ carbon of one sugar and the 5’ sugar of the next, the SP backbone exists due to these phosphodiester bonds In the double-helix model of DNA, the SP backbones are separated by a constant distance, and by Chargaff’s rules, a purine-pyrimidine complementary BP fits into this space, and are held together by hydrogen bonds, 2 for A-T, and 3 for G-C These hydrogen bonds hold the two strands together as well This means that ONE DNA molecules is actually TWO polynucleotide molecules held together by H-bonding Each BP has a length of 0.34 nm alone the long axis of the double helix, and 10 BP make a full turn of the helix molecule, so one full turn of the molecule is 3.4 nm long Two strands of a double helix fit together if they are antiparallel Genetic information is coded into the DNA by the sequence of the nucleotides

12.3 – DNA Replication

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Watson and Crick proposed semiconservative replication o H-bonds break the strands apart, and they unwind and separate o Each strand is a template for the synthesis of its partner o When replication is complete, there are now two helices, each with one strand derived from the parent base-paired with a synthesized one In conservative replication, each of the strands of original DNA serve as a template for a new double helix o After the two complementary copies separate from templates, they wind together into an all new double helix In dispersive replication, both chains of each replicated double helix contain old and new segments Complementary polynucleotide chains to the strands are assembled from individual deoxyribonucleotides by multiple kinds of DNA polymerases (for DNA replication) Deoxyribonucleoside triphosphates (ATP with a deoxyribose sugar linked to a nitrogenous base and 3 phosphate groups) are the substrates required for the polymerization reaction catalyzed by DNA polymerases o Four different deoxyribonucleoside triphosphates are used for each nitrogenous base o They are called dATP, dTTP, dGTP, cCTP The 5’ end of DNA is an exposed PO4 group on the 5’ Carbon; the 3’ end of DNA has an OH group on the 3’ Carbon DNA polymerase can add a nucleotide only to the 3’ end of an existing nucleotide chain, so the template strand is read 3’-5’ direction and the complement is made in the 5’-3’ direction How do DNA Polymerases add nucleotides to the complementary strand? o The template strand and the 3’-OH of the new strand meet at the active site for the polymerization reaction of DNA synthesis, in the “palm” of polymerase o A nucleotide is added to the new strand when a dNTP (dATP, etc…) enters the active site, carrying the complementary base) o By moving along the template strand, DNA polymerase adds to the new strand one nucleotide at a time The sliding DNA clamp is a protein that encircles the DNA and binds to the rear of DNA polymerase, so that the DNA polymerase does not leave the template strand, increasing the rate of DNA synthesis Key Molecular Events of DNA replication: o Two templates unwind

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o DNA polymerase adds nucleotides to an existing chain in the 5’-3’ direction, antiparallel to the template o Nucleotides enter the new chain according to complementary BP rules In semiconservative replication, two strands of the template molecule unwind and expose the template strands The unwinding occurs at a small specific sequence (read 3’-5’) in the bacterial chromosome known as the ORI, where specific proteins bing to the sequence and promote the binding of DNA helicase, which unwinds the molecule to a Yshaped structure called the replication fork – two unwound template strands transitioning to a double-helical DNA SSB proteins (SSBs) coat the exposed single-strands stabilizing the DNA and keeping the two strands from getting back together (SSBs are cockblockers) o SSBs are displaced as the replication enzymes make new polynucleotide chains on the template strands For circular chromosomes (bacteria), unwinding the DNA causes the still wound DNA ahead of the unwinding to become very twisted Topoisomerase prevents still-wound DNA from twisting some more, o by cutting ahead of the replication fork o turning the DNA on one side of the break in the opposite direction of the twisting force and rejoining the two strands Primase synthesizes an RNA primer from the template, then leaves and gives the strand to DNA polymerase which adds to the RNA primer; without the primer there is no replication to begin with; without primase new strands can’t be initiated DNA polymerases synthesize 5’-3’, and since the two strands in a double helix are antiparallel, only one of them runs in a direction that allows DNA polymerase to read 3’-5’ The complementary strand is synthesized continuously in the direction of unwinding, 3’-5’ Discontinuous replication is the replication of the template strands in short lengths that are synthesized in the direction opposite to DNA unwinding The Okazaki fragments are covalently linked into a single chain The new strand synthesized in the direction of unwinding is the leading strand and the strand synthesized opposite direction is the lagging strand The leading strand template runs 3’-5’ and lagging strand template runs 5’-3 DNA polymerase III extends the primer by adding DNA nucleotides

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In the lagging strand, DNA polymerase I removes the RNA primer at the 5’ end of the Okazaki fragment, replacing the RNA nucleotides one by one with DNA ones o RNA nucleotide removal uses the 5’-3’ exonuclease activity, where the primers are digested from its 5’-3’ end o DNA polymerase I stops replacing RNA once it encounters the first DNA nucleotide synthesized in the Okazaki fragment o The DNA base replacing the last RNA base of the primer ends up beside the first DNA base of the Okazaki fragment, and DNA ligase covalently bonds the two Molecular Steps to DNA Replication o DNA helicase unwinds the DNA in 3’-5’ direction o Primases synthesize RNA primers in the 5’-3’ direction of unwinding for leading and opposite for lagging o SSBs bind to the template ahead of the primase to prevent rebinding of the templates o Topoisomerase binds to the double helix ahead of the primase to prevent any more twisting ahead of the replication fork o DNA Polymerase III adds DNA nucleotides to the RNA primer in the 5’3’ direction, and sliding clamp tethers it to the template to improve efficiency o On the lagging strand, primase adds the RNA primer near the point of unwinding and DNA polymerase III adds Okazaki fragments in the 5’-3’ direction away from the ORI o DNA polymerase I removes the RNA primer of the Okazaki fragment through 5’-3’ endonuclease activity and replaces them with DNA nucleotides through 5’-3’ polymerizing activity o DNA ligase covalently bonds the lagging strand fragments Replication advances at 500-1000 nucleotides per second in E. coli and other bacteria, and 50 to 100 per second in eukaryotes An ORI forms a replication bubble and two replication forks going opposite directions RNA primers initiating DNA replication leads to linear chromosomes of eukaryotes getting shorter at each round of replication When the RNA primer is removed from the 3’-5’ template, a gap is left in its place at the 5’ end of the new strand, every where else on the chromosome, these gaps are filled in by DNA polymerase by elongating the 3’ end of a neighbouring nucleotide

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At the very ends of chromosomes, there is no existing nucleotide chain to be elongated, so DNA polymerase can’t fill in the gap with the required DNA nucleotides and the resulting new strand is too short When these new strands become templates, the resulting new chromosomes become even shorter When most somatic cells go through the cell cycle, their chromosomes shorten per division, losing all this DNA can be lethal for the cell and organism Telomeres are noncoding repetitive DNA sequences on the ends of eukaryotic chromosomes that is repeated hundreds to thousands of times o in humans the telomere repeat is 5’-TTAGGG-3’ on the template strand With each replication a fraction of the telomere repeat is lost but the genes are unaffected Telomerase is what maintains the length of telomeres, and it adds DNA to the ends of chromosomes making it a type of DNA polymerase o There will be a single-stranded 5’-3’ region left after RNA removal on the chromosome o Telomerase comes in with its own RNA primer and uses that as template to elongate the 5’-3’ single-strand for several hundred repeats, and the elongated single strand is primed and used as a template as usual o When the RNA primer is removed there will be a single strand like there was at the start Telomerase is not active in somatic cells, so telomeres shorten during cell division, so somatic cells can only divide a certain number of times before they die Telomerase IS normally active in the rapidly dividing cells of the early embryo and germ cells to ensure that chromosomes of gametes have telomeres restored before passing on For cancers, as normal cells become cancer cells, their telomerases are reactivated and cancer cells kind of become immortal, as they can preserve chromosome length through rapid divisions DNA loss doesn’t occur in circular DNA

12.4 – Mechanisms that Correct Replication Errors 

DNA polymerases make very few errors, but the few mistakes that do occur are BP mismatches, which are corrected by either

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o Proofreading mechanisms where the polymerases back up and remove the mispaired nucleotides from the complementary strand  Synthesis only occurs when the most recently added base is complementary to the template strand  If there is a mistake, the polymerase uses its 3’-5’ exonuclease activity to remove the mispaired nucleotide, fixes it, and moves on o Any BP mismatches that remain after proof-reading face correction by DNA repair mechanisms  Correct BP matches fit the 0.34 nm dimensions, so mispaired bases are too large or small to main separation  BP mismatches distort DNA structure, and provide recognition sizes for the repair enzymes  The enzymes look for the mistake, and remove it, and polymerase fills up the gap, and ligase seals it up  The same repair mechanisms fix the effects of radiation The RARE replication errors that remain in DNA after both correction methods are mutations – differences in DNA sequences that appear and remain in replicated copies 12.5 – DNA Organization in Eukaryotic vs Prokaryotic Cells

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Numerous proteins organize the DNA in both eukaryotic and prokaryotic cells in addition to controlling expression In Eukaryotic Chromosomes o Histones are small positively charged proteins complexed with DNA in chromosomes o DNA is wrapped around a core twice consisting of two molecules each (8 molecules total) of histones H2A, H2B, H3, H4 to make one nucleosome o Linker DNA connects adjacent nucleosomes; the length of DNA becomes about 1/7th the length when its wrapped into nucleosomes o Histone H1 binds both to the nucleosomes and the linker DNA, packaging about 6 nucleosomes into a solenoid 30 nm in diameter o This creates a chromatin, which is more resistant to damage than when the DNA is free roaming o Loosely packed regions of chromatin during interphase are euchromatin where genes are active in RNA transcription; densely packed regions are heterochromatin and genes aren’t active o Nonhistones are proteins that help control gene expression

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In Prokaryotes o A closed, circular double helix of DNA packed into the cell nucleoid o Plasmids replicate independently of the host chromosome o Bacterial DNA is organized into loops through interaction with proteins; proteins similar to nonhistones regulate gene activity in prokaryotic organisms Chapter 15 – DNA Technologies and Genomics

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DNA technologies are the techniques used to isolate, purify, analyze and manipulate DNA sequences The use of DNA technologies to manipulate genes for practical purposes is called genetic engineering, a part of biotechnology Biotechnology is simply the use of biological systems for practical purposes, without the use of DNA technology 15.1 – DNA Cloning

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A clone is a line of genetically identical cells or individuals derived from a single ancestor DNA cloning is a method to produce many clones of a piece of DNA, which is a gene of interest Genes are cloned because: o Larger samples to work with o Basic research to find out their biological functions, structure, regulation o Applied research One common method for cloning a gene of interest from a bacterial genome (plasmids)  an isolation method o DNA containing the gene of interest is extracted and cuts it into fragments, one of which contains the gene of interest o Each fragment is inserted into a plasmid producing a collection of recombinant DNA – DNA from two or more different sources joined together o The recombinant DNA is introduced into bacteria, and as the bacterium grows and divides, the recombinant plasmid DNA is also replicated o Then just find the bacterium with the plasmid carrying the gene of interest and isolate for further study The key to DNA cloning is joining two DNA molecules from different sources

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This is made possible by restriction enzymes, which recognize short DNA sequences, called restriction sites, 4-8 BP long and cut the DNA at specific locations within those sequences, restriction fragments are the fragments made after those cuts Restriction refers to their role in bacteria where they cut the DNA molecules of the virus and restrict access, and hiding the restriction sites in the bacterium’s DNA by methylating the bases in the sites Most restriction sites are symmetrical in that the sequence of nucleotides read in the 3’-5’ direction on one strand is the same as that read on the 3’-5’ direction on the complementary strand o the restriction enzyme always recognizes the same sequences as its cut site and cuts at the same place in the sequence Restriction enzymes used in cloning, like EcoRI, cleave the SP backbones of DNA to produce DNA fragments with single-stranded ends These single-stranded ends are sticky ends because the single-stranded regions can form H-bonds with complementary sticky ends on another DNA molecule cut with the same enzyme Recombinant DNA is made in this way; the fragment’s sticky ends are bonded to the plasmid fragment Bacterial plasmids are cloning vectors, molecules that can accept inserted fragments Plasmid cloning vectors are natural plasmids that are engineered to contain two genes useful for the final steps of a cloning experiment for distinguishing bacteria with recombinant plasmids from those that don’t o The ampR gene encodes an enzyme that breaks down ampicillin so the bacteria is resistant to ampicillin o The lacZ+ gene encodes an enzyme part of the lac operon that hydrolyzes lactose

Steps to Clone a Gene of Interest using a Plasmid Cloning Vector and Restriction Enzymes o Isolate genomic DNA containing the gene of interest and cut the genomic DNA into fragments with a restriction enzyme o Cut a circular plasmid vector with the restriction enzyme and the restriction site for the enzyme is within the lacZ+ gene

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o Combine the fragments with the cut plasmids, and seal with DNA ligase; you get a mix of recombinant and nonrecombinant plasmids o Transform the plasmids into E. Coli, some bacteria take up plasmids and some do not o While untransformed bacterium can’t grow on a medium containing ampicillin, transformed bacteria grow on the medium because of the ampR gene on the plasmid o Incubating the plate until colonies that contain the recombinant plasmids appear and are screened The gene of interest has a unique DNA sequence, and through DNA hybridization it is identified when it base-pairs with a complementary DNA/RNA molecule called a nucleic acid probe o If we know the sequence of the gene of interest, we can make the probe to find it o Once the colony containing the plasmids with the gene of interest is identified, the colony can be used to produce large quantities of the cloned gene A collection of clones containing a copy of every DNA sequence in a genome is a genomic library, which is made through cloning vectors To convert single-stranded mRNA to double-stranded DNA for cloning, researchers use reverse transcriptase to make a single-stranded DNA complementary to the mRNA, then degrade the mRNA and use DNA polymerase to make a complementary strand to the first; this creates complementary DNA (cDNA) Polymerase Chain Reaction uses amplification to increase the amount of DNA to point where it can be analyzed or manipulated at a ...