Prokaryotic DNA Replication PDF

CHAPTER 28 DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR PART 1 – PROKARYOTIC DNA REPLICATION  Chapter 28 Heredity “I am the family face Flesh perishes, I live on, Projecting trait and trace Through time to times anon And leaping from place to place over oblivion.” Thomas...

CHAPTER 28 DNA METABOLISM: REPLICATION, RECOMBINATION, AND REPAIR PART 1 – PROKARYOTIC DNA REPLICATION  Chapter 28 Heredity “I am the family face Flesh perishes, I live on, Projecting trait and trace Through time to times anon And leaping from place to place over oblivion.” Thomas Hardy An idealized image of DNA, the substance of heredity.  Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett DNA Replication © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or otherwise on a password-protected website for classroom use.  Essential Questions How is genetic information in the form of DNA replicated? How is the information rearranged? How is its integrity maintained in the face of damage?  Outline How Is DNA replicated? What are the functions of DNA polymerases? Why are there so many DNA polymerases? How is DNA replicated in eukaryotic cells? How are the ends of chromosomes replicated? How are RNA genomes replicated? How is the genetic information rearranged by genetic recombination? Can DNA be repaired? What is the molecular basis of mutation?  The Dawn of Molecular Biology April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." The mechanism: Strand separation, followed by copying of each strand. Each separated strand acts as a template for the synthesis of a new complementary strand.  28.1 How Is DNA Replicated? DNA replication is semiconservative – one of the two original strands is conserved in each progeny molecule DNA replication is bidirectional – it proceeds in both directions from the starting point Replication requires unwinding of the DNA helix DNA replication is semi-discontinuous - the lagging strand is formed from newly synthesized short segments (Okazaki fragments), which are joined to form the final product 28.1 How Is DNA Replicated? Figure 28.1 DNA replication: Strand separation followed by the copying of each strand.  Features of DNA Replication DNA replication is bidirectional Bidirectional replication involves two replication forks, which move in opposite directions DNA replication is semi-discontinuous The leading strand is synthesized continuously The lagging strand is synthesized in segments (Okazaki fragments) which must be joined  Figure 28.2 Bidirectional Replication Comparison of labeling pattern expected during unidirectional versus bidirectional replication. An autoradiogram of E. coli chromosome replication.  Features of Replication First determined in E. coli, but many features are general Replication is bidirectional The double helix must be unwound - by helicases Supercoiling must be overcome - by DNA helicases and gyrases Replication is semi-discontinuous Leading strand is formed continuously Lagging strand is formed from Okazaki fragments - discovered by Tsuneko and Reiji Okazaki – See Figure 28.3 Figure 28.3 The Semidiscontinuous Model for DNA Replication (a) Leading and lagging strand synthesis. Newly synthesized DNA is red. (b) The action of DNA polymerase. dnaB bound to ssDNA DnaB is a hexameric helicase recruited to the replication site and loaded onto the DNA single strand that will become the lagging-strand template during DNA replication  Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett DNA Polymerases © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or otherwise on a password-protected website for classroom use.  28.2 What Are the Functions of DNA Polymerases? The enzymes that replicate DNA are called DNA polymerases All DNA polymerases share the same fundamental catalytic mechanism ✓The incoming base is selected within the polymerase active site, as determined by Watson-Crick geometric interactions ✓Chain growth is in the 5′-3′ direction and is antiparallel to the template strand ✓DNA polymerases cannot initiate DNA synthesis de novo – all require a primer oligonucleotide with a free 3′-OH to build upon  Biochemical Characterization of DNA Polymerases Watson and Crick predicted the existence of an enzyme that makes DNA copies from a DNA template In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA polymerase - DNA polymerase I (Pol I) – in E. coli  Biochemical Characterization of DNA Polymerases DNA polymerases need all four deoxynucleotides, a template and a primer - a single-stranded DNA (with a free 3'-OH) that pairs with the template to form a short double-stranded region The new chain is elongated in the 5′→3′ direction, forming a polynucleotide sequence that is antiparallel and complementary to the template Figure 28.4 The chain elongation reaction catalyzed by DNA polymerase. The 3'-OH carries out a nucleophilic attack on the α- phosphoryl group of the incoming dNTP. PPi is released as a product. The subsequent hydrolysis of PPi by inorganic pyrophosphatase renders the reaction effectively irreversible.  E. coli Cells Have Several Different DNA Polymerases The polymerases of E.coli are compared in Table 28.1 Polymerases I and II, function principally in DNA repair DNA polymerase III is the chief DNA-replicating enzyme of E. coli There are only 40 molecules of Pol III in an E. coli cell 28.2 What Are the Functions of DNA Polymerases? DNA Pol I was discovered in 1957 by Arthur Kornberg and his colleagues. Pol I and Pol II are involved in DNA repair. Pol III is the enzyme responsible for replication of the E. coli chromosome.  DNA Polymerase III The polymerase that carries out replication in E. coli At least 10 different subunits "Core" enzyme has three subunits - α, ε, and θ Alpha (α) subunit is the polymerase Epsilon (ε) subunit is a 3'-exonuclease Theta (θ) subunit is involved in holoenzyme assembly and ε- subunit stabilization The β subunit dimer “sliding clamp” forms a ring around DNA The tau (τ) subunit is for binding the DNA template Enormous processivity - 5 million bases!  DNA Polymerase III The Pol III holoenzyme consists of 17 subunits (αεθ)22β2τ2γδδ′χψ The Composition of E. coli Pol III  Figure 28.5 DNA polymerase III holoenzyme is a dimeric polymerase. One unit of polymerase synthesizes the leading strand, and the other synthesizes the lagging strand  E. Coli Pol III is a Dimeric Polymerase Figure 28.6 (a) Ribbon diagram of the β-subunit dimer of the DNA polymerase III holoenzyme on B-DNA, viewed down the axis of the DNA. One monomer of the β-subunit dimer is blue and the other yellow. (b) Space-filling model of the same structure. The hole formed by the β-subunits is large enough to easily accommodate DNA (diameter approximately 2.5 nm) with no steric repulsion. The rest of Pol III associates with this sliding clamp to form the replicative polymerase. E. Coli Pol III is a Dimeric Polymerase One unit of polymerase synthesizes the leading strand, and the other synthesizes the lagging strand All template strands are read in the 3'-5' direction, so DNA synthesis proceeds in the 5'-3' direction Lagging strand synthesis requires repeated priming E. Coli Pol III is a Dimeric Polymerase Primase (DnaG) bound to the DnaB helicase carries out this priming function, periodically forming new RNA primers on the lagging strand All single-stranded regions of DNA are coated with SSB (single-stranded DNA-binding protein)  A Pol III Holoenzyme Sits at Each Replication Fork The features of the replication fork are shown in Figure below DNA gyrase (topoisomerase) and DnaB helicase unwind the DNA double helix The lagging strand is looped around, and each replicative DNA polymerase moves 5′→3′ relative to its strand, copying template and synthesizing a new DNA strand  A Pol III Holoenzyme Sits at Each Replication Fork Each replicative polymerase is tethered to the DNA by its β2 sliding clamp Downstream on the lagging strand, DNA pol I excises the primer and replaces it with DNA DNA ligase seals the "nicks" between Okazaki fragments DNA Replication in E. coli Requires a Family of Proteins Leading and Lagging Strand Animation DNA Elongation Bidirectional replication  Properties of E. coli DNA Polymerase I Replication occurs 5' to 3' Nucleotides are added at the 3'-end of the strand Pol I catalyzes about 20 cycles of polymerization before the new strand dissociates from template 20 cycles constitutes moderate "processivity" Pol I from E. coli is a 928-amino acid (109-kD) monomer In addition to 5'-3' polymerase, it also has 3'-5' exonuclease and 5'-3' exonuclease activities  3'-Exonuclease Activity of Pol I Removes Nucleotides From the 3'-End of the Chain Why does Pol I have exonuclease activity? The 3'-5' exonuclease activity serves a proofreading function The 3’-exonuclease is a property of the ε-subunit It removes incorrectly matched bases, so that the polymerase can try again The polymerase active site is a proofreader, and the 3’- exonuclease activity is an editor  Figure 28.8 The 3’-5’ exonuclease activity of DNA polymerase I The exonuclease removes nucleotides from the 5’-end of the growing DNA chain. The newly synthesized strand is in purple.  How Does the 5'-Exonuclease Activity of Pol I Accomplish Nick Translation? The 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation" Hans Klenow used trypsin to cleave E. coli pol I between residues 323 and 324, separating 5'-exonuclease (on residues 1-323) and the other two activities (on residues 324-928, the so-called "Klenow fragment”) This 5'-exonuclease activity plays an important role in primer removal during DNA replication  A Mechanism For All Polymerases They require Mg++ Thomas Steitz has suggested that all polymerases use a universal two-metal mechanism. One metal (A) lowers the proton affinity of the 3-O of the growing chain, promoting nucleophilic attack on the α-P of the incoming nucleotide. The second metal (B) assists departure of the product PPi.  28.3 Why Are There So Many DNA Polymerases? Cells have different DNA polymerases for different purposes Polymerases can be grouped in seven functional families, based on sequence homology Family A includes polymerases involved in DNA repair in bacteria Family B includes the eukaryotic polymerases involved in replication of chromosomal DNA Family C is that of the bacterial chromosomal DNA-replicating enzymes Families X and Y act in DNA repair pathways RT designates retrovirus polymerases The Common Architecture of DNA Polymerases The active site lies in a crevice within the palm domain The fingers act in deoxynucleotide recognition and binding The thumb is responsible for DNA binding Figure 28.10 A structural paradigm for DNA polymerases, bacteriophage RB69 DNA polymerase.  Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett UP NEXT DNA Replication in EUKARYOTES © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or otherwise on a password-protected website for classroom use.