DNA Structure, Replication, and Manipulation - Genetics - 2024 PDF

Chapters 6 DNA Structure, Replication, and Chapter 6 Manipulation  Agenda DNA Structure Replication 2  Conception Check Provide two characteristics of DNA that we discussed in Chapter 1...

Chapters 6 DNA Structure, Replication, and Chapter 6 Manipulation  Agenda DNA Structure Replication 2  Conception Check Provide two characteristics of DNA that we discussed in Chapter 1 3  Genomes and DNA The genetic complement of a cell or virus constitutes its genome. – commonly used to refer to one complete haploid set of chromosomes (sperm or egg) C-value: DNA content of the haploid genome.  Genomes and DNA The genetic complement of a cell or virus constitutes its genome. – commonly used to refer to one complete haploid set of chromosomes (sperm or egg) C-value: DNA content of the haploid genome. 2 typical units of length of nucleic acids: – Kilobase (kb) 103 base pairs – Megabase (Mb) 106 base pairs  The C-Value Paradox Genome size among species of: – protozoa differs 5800-fold – arthropods differs 250-fold – fish differs 350-fold – algae differs 5000-fold – angiosperms differs 1000-fold.  The C-Value Paradox Genome size among species of protozoa differs by 5800-fold, among arthropods by 250-fold, fish 350-fold, algae 5000-fold, and angiosperms 1000-fold. The C-value paradox: Among eukaryotes, there is no consistent relationship between the C-value and the metabolic, developmental, or behavioral complexity of the organism. – in higher organisms, much of the DNA has functions other than coding for the amino acid sequence of proteins.  Purines and Pyrimidines Two of the bases, A and G, have a double-ring structure; these are called purines. The other two bases, T and C, have a single-ring structure; these are called pyrimidines.  Nucleotides DNA is a polymer of four deoxyribonucleotides. Nucleotides are composed of: – 2'-deoxyribose (a five-carbon sugar), – phosphoric acid, – the four nitrogen-containing bases denoted A, T, G, and C.  Polynucleotide Chains The nucleotides are joined to form a polynucleotide chain by phosphodiester bonds. – phosphate attached to the 5' carbon of one sugar is linked to the hydroxyl group attached to the 3' carbon of the next sugar in line.  Polynucleotide Chains The nucleotides are joined to form a polynucleotide chain by phosphodiester bonds. – phosphate attached to the 5' carbon of one sugar is linked to the hydroxyl group attached to the 3' carbon of the next sugar in line.  The Double Helix DNA is a right-handed helix – two polynucleotide chains twisted around one another Nucleotides pair with H bonds: – Adenine pairs with thymine – Guanine with cytosine.  The Double Helix DNA is a right-handed helix – two polynucleotide chains twisted around one another Nucleotides pair with H bonds: – Adenine pairs with thymine – Guanine with cytosine. The stacking of the base pairs on top of one another helps hold the strands together.  DNA Structure The backbone of each strand – deoxyribose sugars alternating with phosphate groups that link the 5' carbon of one sugar to the 3' carbon of the next sugar in line. The paired strands are said to be antiparallel. DNA molecule showing the antiparallel orientation of the complementary strands.  DNA: Watson-Crick Model 3-D structure of the DNA molecule: – Double helix forms major and minor grooves. – Diameter of the helix = 20 Angstroms. – Each turn of the helix = 10 bases = 34 Angstroms.  Conception Check 1. Which chemical group is at the 5' end of a single polynucleotide strand? A) Hydroxyl group B) Diester group C) Purine base D) Phosphate group E) Nitrogen group  Conception Check 1. Which chemical group is at the 5' end of a single polynucleotide strand? A) Hydroxyl group B) Diester group C) Purine base D) Phosphate group E) Nitrogen group Ans: D  Chapters 6 DNA Structure, Replication, and Chapter 6 Manipulation  Agenda Last Class: DNA Structure Today: Replication 19  Nucleic Acid Structure Summary: DNA is the genetic material of cellular life The monomers of DNA are nucleotides DNA nucleotides base pair Right-handed double helix model  Nucleic Acid Structure Summary: DNA is the genetic material of cellular life The monomers of DNA are nucleotides DNA nucleotides base pair Right-handed double helix model Strands are anti-parallel and held together by H bonds and hydrophobic interactions Helix has major and minor grooves and uniform diameter The structure of DNA suggests the mode of DNA replication  The Watson-Crick Model of Replication Hydrogen bonds between DNA bases break to allow strand separation. Each DNA strand is a template for the synthesis of a new strand.  The Watson-Crick Model of Replication Hydrogen bonds between DNA bases break to allow strand separation. Each DNA strand is a template for the synthesis of a new strand. Template (parental) strand determines the sequence of bases in the new strand (daughter) by complementary base pairing. Replication is semiconservative.  Results of the Meselson Stahl Experiment Are Consistent with Semiconservative Replication  Results of the Meselson Stahl Experiment Are Consistent with Semiconservative Replication E coli grown in 15N CsCl solution = gel - Separates by size  Results of the Meselson Stahl Experiment Are Consistent with Semiconservative Replication  Results of the Meselson Stahl Experiment Are Consistent with Semiconservative Replication  Results of the Meselson Stahl Experiment Are Consistent with Semiconservative Replication  Critical Thinking Check How do these result prove replication is "semiconservative." 29  Chapters 6 DNA Structure, Replication, and Chapter 6 Manipulation  Agenda Types of Replication Replication Mechanism Telomerase *Manipulation 31  Conception Check Distinguish the Purines from Pyrimidines found in DNA and describe their structure. 32  Purines and Pyrimidines Two of the bases, A and G, have a double-ring structure; these are called purines. The other two bases, T and C, have a single-ring structure; these are called pyrimidines.  Types of Replication Theta (ᶿ) Replication (bacterial) – Bidirectional – Unidirectional Rolling Circle Multiple initiation – Bidirectional  Circular (Bacterial) DNA Replication Autoradiogram of the intact replicating circular chromosome of E. coli shows that: – DNA synthesis is bidirectional. – Replication starts from a single site called origin of replication (OR). The region in which parental strands are separating and new strands are being synthesized is called a replication fork. Bidirectional Theta Replication Unidirectional Theta Replication Theta Replication  Rolling Circle Replication Some bacteria and eukaryotic viruses One DNA strand is cut by a nuclease to produce a 3'-OH extended by DNA polymerase.  Rolling Circle Replication Some bacteria and eukaryotic viruses One DNA strand is cut by a nuclease to produce a 3'-OH extended by DNA polymerase. New strand displaces from the template strand as DNA synthesis continues. The displaced strand is a template for a complementary DNA strand.  Replication of Chromosomal DNA in Eukaryotes The linear DNA duplex in a eukaryotic chromosome also replicates bidirectionally. Replication is initiated at many sites along the DNA. Multiple initiation is a means of reducing the total replication time.  Replication of Chromosomal DNA in Eukaryotes Origins of replication are about 40,000 bp apart – chromosome to be replicated in 15 to 30 minutes. Usually takes from 5 to 10 hours. – Not simultaneous  Conception Check What is the major difference between unidirectional and bidirectional theta replication?  Conception Check What is the major difference between unidirectional and bidirectional theta replication?  Conception Check What direction is DNA synthesized? 45 Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase Single-strand binding protein (SSB) Gyrase (topoisomerase II) Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase: unwinds DNA at replication fork separate the parental strands. Single-strand binding protein (SSB) Gyrase (topoisomerase II) Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase unwinds DNA at replication fork to separate the parental strands. Single-strand binding protein (SSB) stabilizes single strands of DNA at replication fork. – prevents DNA from rebinding Gyrase (topoisomerase II) Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase unwinds DNA at replication fork to separate the parental strands. Single-strand binding protein (SSB) stabilizes single strands of DNA at replication fork. Gyrase (topoisomerase II) introduces a double-stranded break ahead of the replication fork and swivels the cleaved ends to relieve the stress of the helix unwinding. Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase unwinds DNA at replication fork to separate the parental strands. Single-strand binding protein (SSB) stabilizes single strands of DNA at replication fork. Gyrase (topoisomerase II) introduces a double-stranded break ahead of the replication fork and swivels the cleaved ends to relieve the stress of the helix unwinding. ?  DNA Synthesis Synthesis can only be done 5’ to 3’ Results in 2 types of Strands during synthesis: – Leading Strand – Lagging Strand  DNA Synthesis Synthesis can only be done 5’ to 3’ Results in 2 types of Strands during synthesis: – Leading strand - synthesized continuously – Lagging strand - made in small precursor fragments (Okazaki fragments)  DNA Synthesis Synthesis can only be done 5’ to 3’ Results in 2 types of Strands during synthesis: – Leading strand - synthesized continuously – Lagging strand - made in small precursor fragments (Okazaki fragments) o 1000–2000 base pairs in prokaryotic cells o 100–200 base pairs in eukaryotic cells.  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) DNA polymerase  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) initiates strand synthesis by forming RNA primer. DNA polymerase  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) initiates strand synthesis by forming RNA primer. DNA polymerase forms the phosphodiester bond between adjacent nucleotides in a new DNA acid chain in the 5' to 3' direction.  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) initiates strand synthesis by forming RNA primer. DNA polymerase forms the phosphodiester bond between adjacent nucleotides in a new DNA acid chain in the 5' to 3' direction. – proofreading function that corrects errors in replication. o Only the last nucleotide  Requirements for Completing Lagging Strand Prokaryotes 2 enzymes needed to finish lagging strand: DNA polymerase I DNA ligase  Requirements for Completing Lagging Strand Prokaryotes 2 enzymes needed to finish lagging strand: DNA polymerase I – Primer removal: 5’  3' exonuclease activity o One nucleotide at a time – Replacement of RNA with DNA DNA ligase  Requirements for Completing Lagging Strand Prokaryotes 2 enzymes needed to finish lagging strand: DNA polymerase I – Primer removal: 5’  3' exonuclease activity – Replacement of RNA with DNA DNA ligase – Sealing nicks in the backbone  Requirements for Completing Lagging Strand Eukaryotes 2 enzymes needed to finish lagging strand: DNA polymerase delta DNA ligase  Requirements for Completing Lagging Strand Eukaryotes 2 enzymes needed to finish lagging strand: DNA polymerase delta – Primer removal: 5’  3' exonuclease activity o All at once – Replacement of RNA with DNA DNA ligase – Sealing nicks in the backbone  Chapter 6 DNA Structure, Replication, and Chapter 6 Manipulation  Conception Check What direction is DNA synthesized? 64  Agenda DNA Replication/Synthesis Telomerase Manipulation 65  Types of Replication Theta (ᶿ) Replication (bacterial) – Bidirectional – Unidirectional Rolling Circle Multiple initiation – Bidirectional  Circular (Bacterial) DNA Replication Autoradiogram of the intact replicating circular chromosome of E. coli shows that: – DNA synthesis is bidirectional. – Replication starts from a single site called origin of replication (OR). The region in which parental strands are separating and new strands are being synthesized is called a replication fork. Bidirectional Theta Replication Unidirectional Theta Replication Theta Replication  Rolling Circle Replication Some bacteria and eukaryotic viruses One DNA strand is cut by a nuclease to produce a 3'-OH extended by DNA polymerase.  Rolling Circle Replication Some bacteria and eukaryotic viruses One DNA strand is cut by a nuclease to produce a 3'-OH extended by DNA polymerase. New strand displaces from the template strand as DNA synthesis continues. The displaced strand is a template for a complementary DNA strand.  Replication of Chromosomal DNA in Eukaryotes The linear DNA duplex in a eukaryotic chromosome also replicates bidirectionally. Replication is initiated at many sites along the DNA. Multiple initiation is a means of reducing the total replication time.  Replication of Chromosomal DNA in Eukaryotes Origins of replication are about 40,000 bp apart – chromosome to be replicated in 15 to 30 minutes. Usually takes from 5 to 10 hours. – Not simultaneous  Conception Check What is the major difference between unidirectional and bidirectional theta replication?  Conception Check What is the major difference between unidirectional and bidirectional theta replication?  DNA Replication 3 stages 1. Initiation 2. Priming & Synthesis/Extension 3. Ligation/Completion Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase Single-strand binding protein (SSB) Gyrase (topoisomerase II) Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase: unwinds DNA at replication fork separate the parental strands. Single-strand binding protein (SSB) Gyrase (topoisomerase II) Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase unwinds DNA at replication fork to separate the parental strands. Single-strand binding protein (SSB) stabilizes single strands of DNA at replication fork. – prevents DNA from rebinding Gyrase (topoisomerase II) Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase unwinds DNA at replication fork to separate the parental strands. Single-strand binding protein (SSB) stabilizes single strands of DNA at replication fork. Gyrase (topoisomerase II) introduces a double-stranded break ahead of the replication fork and swivels the cleaved ends to relieve the stress of the helix unwinding. Proteins Interacting with DNA During Replication 3 protein help initiate replication: Helicase unwinds DNA at replication fork to separate the parental strands. Single-strand binding protein (SSB) stabilizes single strands of DNA at replication fork. Gyrase (topoisomerase II) introduces a double-stranded break ahead of the replication fork and swivels the cleaved ends to relieve the stress of the helix unwinding. ?  DNA Synthesis Synthesis can only be done 5’ to 3’ Results in 2 types of Strands during synthesis: – Leading Strand – Lagging Strand  DNA Synthesis Synthesis can only be done 5’ to 3’ Results in 2 types of Strands during synthesis: – Leading strand - synthesized continuously – Lagging strand - made in small precursor fragments (Okazaki fragments)  DNA Synthesis Synthesis can only be done 5’ to 3’ Results in 2 types of Strands during synthesis: – Leading strand - synthesized continuously – Lagging strand - made in small precursor fragments (Okazaki fragments) o 1000–2000 base pairs in prokaryotic cells o 100–200 base pairs in eukaryotic cells.  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) DNA polymerase  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) initiates strand synthesis by forming RNA primer. DNA polymerase  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) initiates strand synthesis by forming RNA primer. DNA polymerase forms the phosphodiester bond between adjacent nucleotides in a new DNA acid chain in the 5' to 3' direction.  Priming and Synthesis 3 enzymes assist with priming/synthesis: Primosome (RNA primase complex) initiates strand synthesis by forming RNA primer. DNA polymerase forms the phosphodiester bond between adjacent nucleotides in a new DNA acid chain in the 5' to 3' direction. – proofreading function that corrects errors in replication. o Only the last nucleotide  Requirements for Completing Lagging Strand Prokaryotes 2 enzymes needed to finish lagging strand: DNA polymerase I DNA ligase  Requirements for Completing Lagging Strand Prokaryotes 2 enzymes needed to finish lagging strand: DNA polymerase I – Primer removal: 5’  3' exonuclease activity o One nucleotide at a time – Replacement of RNA with DNA DNA ligase  Requirements for Completing Lagging Strand Prokaryotes 2 enzymes needed to finish lagging strand: DNA polymerase I – Primer removal: 5’  3' exonuclease activity – Replacement of RNA with DNA DNA ligase – Sealing nicks in the backbone  Requirements for Completing Lagging Strand Eukaryotes 2 enzymes needed to finish lagging strand: DNA polymerase delta DNA ligase  Requirements for Completing Lagging Strand Eukaryotes 2 enzymes needed to finish lagging strand: DNA polymerase delta – Primer removal: 5’  3' exonuclease activity o All at once – Replacement of RNA with DNA DNA ligase – Sealing nicks in the backbone 95 96  Requirements for Completing Lagging Strand Eukaryotes 2 enzymes needed to finish lagging strand: DNA polymerase delta – Primer removal: 5’  3' exonuclease activity o All at once – Replacement of RNA with DNA DNA ligase – Sealing nicks in the backbone  Last Question Muddiest Point? 98  Chapters 6 DNA Structure, Replication, and Chapter 6 Manipulation  Agenda Review Replication Telomerase DNA manipulation 100  Conception Check What is the function of the following enzymes: Helicase Single-strand binding protein (SSB) Gyrase (topoisomerase II) Primosome (RNA primase complex) DNA polymerase I DNA polymerase delta DNA ligase Leading and Lagging Strand Synthesis 3 4 5 6* 1 2 5  Coordination of Leading and Lagging Strand Synthesis Leading and lagging strands are synthesized at the same time Loop forms (trombone model)  Conception Check What is the telomere and where are they located? 104 105 106  Telomerase Telomerase is not active in most somatic cells of animals (maybe not all animals??) Telomere region is shortened with each cell division – Cells eventually cannot reproduce Stem cells and malignant cells do have telomerase activity 107  Restriction Enzyme Function Restriction enzymes – cleave duplex DNA at particular nucleotide sequences. – Recognize specific nucleotide sequences for cleavage (restriction site) o the restriction site normally reads the same on both strands.  Restriction Enzyme Cleavage Symmetric vs asymmetric cuts: Asymmetric cuts – Results in overhanging nucleotides – Sticky ends Symmetric cuts – at the same site in both strands. – blunt ends.  Restriction Digestion of DNA sequence specificity: – a particular restriction enzyme produces a unique set of restriction fragments for a particular DNA molecule. – Another enzyme will produce a different set of restriction fragments from the same DNA molecule. A map showing the unique sites of cutting of a particular DNA molecule by restriction enzyme is called a restriction map.  Conception Check What are the differences (cause & appearance) between sticky and blunt end? 111  PCR Polymerase Chain Reaction 112  The Polymerase Chain Reaction Polymerase chain reaction (PCR) – amplifies a particular DNA fragment.  The Polymerase Chain Reaction Polymerase chain reaction (PCR) – amplifies a particular DNA fragment. Primers – complementary – Bind to the ends of the target sequence – The number of copies doubles in each round o eventually overwhelming any other sequences that may be present.  PCR Thermocycling 3 Steps: 1. Denaturation (95oC) 2. Reannealing (50-60oC) 3. Elongation (72oC) Repeat steps 1–3 20–30 times.  Taq Polymerase DNA polymerase isolated from Thermus aquaticus, an inhabitant of hot springs Functions at 72oC Remains active at 95oC DNA Sequence Analysis  DNA Sequence Analysis DNA sequence analysis determines the order of bases in DNA. – Aka Sanger Sequencing The dideoxy sequencing method – DNA synthesis in the presence of small amounts of fluorescently labeled nucleotides that contain the sugar dideoxyribose instead of deoxyribose.  When Dideoxynucleotides Are Incorporated dideoxyribose = termination Dideoxy method of DNA sequencing.  When Dideoxynucleotides Are Incorporated dideoxyribose = termination The products of DNA synthesis are then separated by electrophoresis. In principle, the sequence can be read directly from the gel.  Interpreting the Results Each molecule produces one base longer than the previous band. Each dideoxynucleotide is labeled by different fluorescent dye. – G, black; A, green; T, red; C, purple. As each band comes off the bottom of the gel, the fluorescent dye that it contains is determined.  Conception Check What were the three steps of PCR? 122  Conception Check What were the three steps of PCR? 3 Steps: 1. Denaturation (95oC) 2. Reannealing (50-60oC) 3. Elongation (72oC) Repeat steps 1–3 20–30 times. 123  Next Class Exam 2 124  Last Question Muddiest Point? 125