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Heredity Chapter 11

Terms in this set (42)

DNA as a Template
DNA strands serve as template
-Arrangement and nature of nitrogenous bases allow DNA strands to serve as templates
-Complementarity of DNA strands allows each strand to serve as template for synthesis of the other
Three Modes of Replication
Three modes of DNA replication:

Semiconservative
-Each replicated DNA molecule consists of one "old" and one new strand

Conservative
-Two newly synthesized strands come together - original helix is conserved

Dispersive
-Parental strands are dispersed into two new double helices
Meselson-Stahl Experiment
Meselson and Stahl (1958)
-15N-labeled E. coli grown in medium containing 14N
-Each new DNA molecule consists of one old and one newly synthesized strand
-Provided strong evidence that DNA is semiconservative in prokaryotes
Semiconservative DNA in Eukaryotes
Taylor-Woods-Hughes (1957)
-Vicia faba (broad bean) was used to demonstrate DNA replication is semiconservative in eukaryotes
-Monitored process of replication with labeled 3H-thymidine and performed audioradiography
Audiography
-Pinpoints location of radioisotope in cell
-Photographic emulsion placed over cellular material and stored in the dark
-Develops much like photographic film
-Result: Presence of dark grains identifies location of newly synthesized DNA
Origins, Forks, and Units of Replication
DNA replication
-DNA replication begins at the ORI (origin of replication)
-At site of replication, helix is unwound, creating replication fork
-Replication is bidirectional; therefore, there are two replication forks
-Replicon: Length of DNA replicated
Bacterial Replication
Bacteria have only one ORI
-Single circular DNA
-DNA synthesis originates at OriC
-E. coli replicon consists of entire genome (4.6 million base pairs)
DNA Synthesis in Bacteria
DNA synthesis in bacteria involves five polymerases (DNA Pol)
-DNA polymerase I
-DNA polymerase II
-DNA polymerase III
-DNA polymerase IV
-DNA polymerase V
DNA Polymerase I
DNA polymerase I
-Isolated enzyme from E. coli
-Enzyme directs DNA synthesis
-Requires DNA template and all four deoxyribonucleoside triphosphates (dNTPs)
-Enzyme consists of polypeptide with 928 amino acids
Chain Elongation
Chain elongation by DNA polymerase I
-Occurs in 5' to 3' direction by adding one nucleotide at a time to 3' end
-Nucleotide added, two terminal phosphates cleaved off, providing newly exposed 3'-OH
-3'-OH can participate in addition of another nucleotide as DNA synthesis proceeds
DNA Pol I, II, and III
DNA Pol I, II, and III
-Can elongate existing DNA strand (primer)
-Cannot initiate DNA synthesis

Exonuclease activity 3'-5'
-All three possess 3' to 5' exonuclease activity: proofread newly synthesized DNA, remove/replace incorrect nucleotides

Exonuclease activity 5'-3'
-Only DNA polymerase I
-Excises primers - fills in gaps left behind
DNA Repair
DNA polymerases I, II, IV, and V
-Involved in various aspects of DNA repair
-Repair DNA damaged by external forces such as UV light
DNA Pol III Holoenzyme
DNA polymerase III
-Holoenzyme: Active form of DNA Pol III
-Holoenzyme contains core enzyme complexes made up of subunits
-Subunits each have separate functions
+ α- 5'-3' polymerization
+ ε- 3'-5' exonuclease
+ θ- Core assembly
DNA Replication Issues
Seven key issues must be resolved during DNA replication:
-Unwinding of helix
-Reduce increased coiling generated during unwinding
-Synthesis of primer for initiation
-Discontinuous synthesis of second strand
-Removal of the RNA primers
-Joining of gap-filling DNA to adjacent strand
-Proofreading
DnaA - Unwinding the Helix
DnaA
-Initiator protein encoded by dnaA gene
-Binds to ORI causing conformation change
-Causes helix to destabilize and open up
-Exposes ssDNA
DNA Helicase
-Made of DnaB polypeptides
-Hexamer of subunits: Assembles around exposed ssDNA
-Subsequently recruits holoenzyme to bind replication fork and initiate replication
-Helicases require energy supplied by hydrolysis of ATP - denatures hydrogen bonds and stabilizes double helix
Single-Stranded Binding Proteins (SSBPs)
-Stabilize the open conformation of helix
-Bind specifically to single strands of DNA
Supercoiling
DNA gyrase
-Enzyme relieves coiled tension from unwinding of helix (DNA supercoiling)
-Member of larger enzyme group: DNA topoisomerases
-Makes single- or double-stranded cuts
-Driven by energy released during ATP hydrolysis
RNA Polymerase: Primase
Primase: RNA polymerase
-Recruited to replication form by helicase
-Synthesizes RNA primer
-Provides free 3'-OH required by DNA polymerase III for elongation
RNA Priming
DNA polymerase I
-Removes primer and replaces it with DNA

RNA priming
-Universal phenomenon
-Found in bacteria, viruses, and several eukaryotic organisms
Continuous and Discontinuous DNA Synthesis
Two strands of double helix are antiparallel: 5'-3' and 3'-5'
-DNA Pol III ONLY synthesizes 5'-3'

Continuous DNA synthesis
-Leading strand

Discontinuous DNA synthesis
-Lagging strand
Okazaki Fragments
Okazaki fragments
-Lagging strand synthesized as Okazaki fragments, each with RNA primer

DNA polymerase I
-Removes primers on lagging strand

DNA ligase
-Catalyzes formation of phosphodiester bonds
-Seals nicks and joins fragments
Concurrent Synthesis
Both DNA strands synthesized concurrently
-Concurrent DNA synthesis achieved on both strands at single replication fork
-Lagging strand is looped
Inverts physical but not biochemical direction
-DNA clamp prevents core enzyme dissociation from template
Proofreading
Proofreading and error correction
-Integral part of DNA replication
-DNA polymerase not always perfect
-Synthesis of noncomplementary base pairs inserted occasionally
-DNA polymerase exonuclease activity of 3'-5' allows for excise of nucleotides
Enzymes and Proteins are Essential to DNA Synthesis
-DNA polymerase III core enzymes
-SSBPs: single-stranded binding proteins
-DNA gyrase
-DNA helicase
-RNA primers
Replication Controlled by Variety of Genes
Mutations
-Interrupt or impair aspects of replication
-Examples: Lethal mutations
+Ligase-deficient mutations
+Proofreading-deficient mutations
-Conditional mutations: Expressed under specific conditions
Temperature-Sensitive Mutation
-Example of conditional mutation
-May not be expressed at particular permissive temperature
-Mutant cells grown at restrictive temperature and mutant phenotype expressed
Eukaryotic DNA Replication
Eukaryotic and bacterial DNA replication shares many features
-Double-stranded DNA unwound at ORI
-Replication forks formed
-Bidirectional synthesis creates leading and lagging strands
-Eukaryotic polymerases require four deoxyribonucleoside triphosphates, template, and primer
Eukaryotic DNA Replication is More Complex
-More DNA than prokaryotic cells
-Linear chromosomes
-DNA complexed with nucleosomes
Eukaryotic Replication: Multiple ORIs
-Eukaryotic chromosomes contain multiple ORIs
-Facilitates rapid synthesis of large quantity of DNA
ARSs
Yeast genomes contain 250-400 origins

Yeast ORI:
-Autonomously replicating sequences (ARSs)
-120 base pairs of consensus sequences
-Consensus sequence: Sequence that is the same in all yeast ARSs
Prereplication Complex
Eukaryotic ORIs control timing of DNA replication

Prereplication complex (pre-RC)
-Assembles at replication ORIs
-Early G1 phase of cell cycle:
+Origin recognition complex (ORC) recognizes ORI and tags ORI as site of initiation
Multiple Eukaryotic DNA Polymerases
DNA polymerases involved in nuclear genome DNA replication
-Poly α, δ, and ε: Involved in initiation and elongation
-Pol α
-Possesses low processivity
-RNA primer synthesis during initiation on leading and lagging strands
Polymerase Switching
Polymerase switching occurs once the primer is in place
-Pol α and ε replaced by Pol δ for elongation
-Pol δ synthesizes lagging strand
-Pol ε synthesizes leading strand
Replication through Chromatin
Eukaryotic DNA complexed with binding proteins (chromatin)

200 base pair nucleosomes wrap around eight histone proteins
-Nucleosomes must be stripped away before polymerase can begin synthesis
Telomeres
-Inert chromosomal ends that protect intact eukaryotic chromosomes from improper fusion or degradation
-Long stretches of short repeating sequences preserve the integrity/stability of chromosomes
-Eukaryotic enzyme
-Ribonucleoprotein: RNA serves as template for synthesis of DNA complement (reverse transcriptase)
-Once RNA primer removed on lagging strand, no free 3'-OH to elongate
-Telomerase adds repeats of six-nulceotide sequence to 3' end to fill gaps
Telomerase Activity
Telomeres of chromosomes shorten with each cell division
-In most eukaryotic somatic cells, telomerase is not active

Stem cells and malignant cells maintain telomerase activity - immortalized

Telomerase activity and telomere length linked to aging, cancer, and other diseases
Genetic Recombination
-Homologous recombination: Genetic exchange at equivalent positions along two chromosomes with substantial sequence homology
DNA Recombination, Like DNA Replication, Is Directed by Specific Enzymes
Genetic recombination involves:
-Endonuclease nicking
-Strand displacement and pairing with complement
ligation
-Branch migration
-Duplex separation - generates characteristic Holiday structure
Enzymes and Proteins
Enzymes and proteins involved in homologous recombination
-RecA protein in E. coli (Rad51 in eukaryotic cells) promotes exchange of reciprocal ssDNA molecules
-Bring about strand invasion and displacement
Gene Conversion
Gene conversion
-Consequence of homologous recombination
-Characterized by nonreciprocal genetic exchange between two DNA molecules
-Can occur in somatic cells during DNA repair

-Region of DNA within heteroduplex
-Results in hybrid dsDNA
Meselson-Stahl Experiment
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