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Chapt 20 DNA Replication I: Basic Mechanism and Enyzmology


Chapt 20 DNA Replication I: Basic Mechanism and Enyzmology Student learning outcomes: Describe general features of semi-conservative DNA replication: leading, lagging ... – PowerPoint PPT presentation

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Title: Chapt 20 DNA Replication I: Basic Mechanism and Enyzmology

Chapt 20 DNA Replication I Basic Mechanism and
  • Student learning outcomes
  • Describe general features of semi-conservative
    DNA replication leading, lagging, strands
    requirement for primers bidirectional, rolling
  • Describe DNA polymerases general enzymology and
    comparison of prokaryotes, eukaryotes
  • Describe major types of DNA damage and repair
  • Important Figures 1, 4, 7, 9, 10, 13, 14, 15,
    16, 18, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33,
    34, 36, 37, 38, 39, 40, 41
  • Review problems 8-11, 13-15, 22-24, 27-29,
    32-35 AQ4

20.1 General Features of DNA Replication
  • Double helical model for DNA complementary
  • Each strand is template for new partner strand
  • Semiconservative model for DNA replication 5 -gt
  • Leading strand continuous synthesis
  • Half-discontinuous (short pieces on lagging
    strand are later stitched together)
  • Requires RNA primers
  • Usually bidirectional (bacterial and eukaryotes)
  • Origin of replication (ori) fixed starting point
  • Replicon DNA under control of one ori

Semi-discontinuous Replication
  • DNA polymerase only synthesizes 5?3 direction
  • RNA primers 10-12 nt long in E. coli
  • Leading strand replicates continuously in
    direction of movement of fork
  • Lagging strand replicates discontinuously in
    direction opposite to fork (as 1-2 kb Okazaki

Fig. 7
Bidirectional Replication
  • Replication structure resembles Greek letter, ?
  • DNA replication begins with creation of bubble
    small region where parental strands separated,
    progeny DNA synthesized
  • As bubble expands, replicating DNA is ? shape

Fig. 9
Rolling Circle Replication
  • Circular DNAs can replicate as rolling circle
  • One strand of dsDNA is nicked, 3-end extended
  • Uses intact DNA strand as template
  • 5-end gets displaced lagging synthesis fills in
  • Phage l leading strand elongates continuously
  • displaced strand serves as template for
    discontinuous, lagging strand synthesis can get
    many genome sized piece

Fig. 20
20.2 Enzymology of DNA Replication
  • gt30 different polypeptides to replicate E. coli
  • Biochemical purifications, conditional mutants to
    examine activities of proteins (essential
  • DNA polymerases enzymes that make DNA
  • Require primer, Mg, buffer, dNTPs
  • 3 DNA polymerases in E. coli
  • pol I - repair enzyme
  • pol II - non-essential
  • pol III the real replication enzyme

DNA Polymerase I
Fig. 16
  • E. coli DNA polymerase I
  • first enzyme (1958, Arthur Kornberg)
  • Pol I has 3 distinct activities
  • DNA polymerase
  • 3?5 exonuclease proofreading
  • 5?3 exonuclease degrades strand ahead of it
  • Can remove primers
  • Mild proteolytic treatment -gt2 polypeptides
  • Klenow fragment (lacks 5-gt3 exonuclease)
  • Can fill in sticky ends left by restriction
    enzymes (Fig. 15)
  • Steitz structure 1987

Pol III Holoenzyme
Pol III core has 3 subunits catalysis,
proofread, Pol III g complex has 5 subunits
DNA-dependent ATPase Pol III holoenzyme includes
b subunit
Charles McHenry (CU SOM) biochemical studies
Eukaryotes have multiple DNA Polymerases
  • Mammalian cells 5 different DNA polymerases
  • Polymerases d and a replicate both DNA strands
  • PCNA factor helps with processivity

Other enzymes for replication E. coli
  • Helicase uses ATP to unwind strands
  • Creates positive supercoils
  • dnaB gene
  • Single strand DNA binding protein SSB
  • Stimulates polymerization
  • DNA gyrase (topoisomerase II)
  • Negative supercoils, swivel

20.3 DNA Damage and Repair
  • DNA can be damaged in many different ways
  • Cells have many ways to repair damage, easier to
    repair before DNA is replicated
  • if unrepaired, damage can lead to mutation
  • DNA damage is not the same as mutation, but it
    can lead to mutation
  • DNA damage is chemical alteration
  • Mutation is inherited change in base pair
  • Common examples of DNA damage
  • Base modifications caused by alkylating agents
  • Pyrimidine dimers caused by UV radiation

Alkylation of Bases Causes Damage
  • Alkylation - process where electrophiles
  • Attack negative centers
  • Add carbon-containing groups (alkyl groups)
  • Common targets (red) N7 of G, N3 of A,
    phosphodiester bond
  • O6 of G

Fig. 27
Alkylation of Bases Causes Damage
  • Alkylating agents like ethylmethane sulfonate
  • Some alkylations dont change base-pairing
  • Others cause DNA replication to stall
  • Cytotoxic
  • Mutations if cell attempts to replicate without
  • Others change base-pairing properties, so are
  • Ethyl O6-G mispairs with T -gt GC -gtAT transition

Fig. 28
UV Radiation Damages DNA
  • Ultraviolet rays (260 nm)
  • Comparatively low energy
  • Moderate type of damage
  • Result in formation of pyrimidine dimers
  • Mostly T-T dimers
  • T-T dimers distort DNA, block replication and

Fig. 29 Thymine dimers have cyclobutane ring
Ionizing Radiation Damages DNA
  • Gamma and x-rays
  • Much more energetic
  • Ionize molecules around DNA
  • Highly reactive free radicals attack DNA
  • Alter bases
  • Break DNA strands
  • Especially double strand
  • (useful cancer therapy)

Fig. 30 oxidative damage forms 8-oxo-guanine At
replication, A often is inserted opposite -gt
UV DNA Damage can be directly reversed
  • Photoreactivation (light repair)
  • DNA photolyase uses energy from near-UV to blue
    light to break bonds holding 2 pyrimidines
  • Enzyme in most organisms (not placental mammals)

Fig. 31
Reversing High Energy DNA Damage
  • O6 alkylations on G residues directly reversed by
    enzyme O6-methylguanine methyltransferase
  • Enzyme accepts alkyl group onto SH of Cys - and
    is inactivated (suicide enzyme)
  • In E. coli, enzyme is induced by DNA alkylation

Fig. 32
Excision Repair
  • Only small percentage of DNA damage products are
    directly reversed
  • Excision repair removes most damaged nucleotides
  • Damaged DNA is removed
  • Replaced with fresh DNA
  • Both base and nucleotide excision repair

Base Excision Repair (BER) specific enzymes
remove damaged base
  • DNA glycosylase
  • Extrudes base in damaged base pair, and clips out
  • Leaves apurinic or apyrimidinic (AP) site that
    attracts DNA repair enzymes
  • DNA repair enzymes
  • Remove remaining deoxyribose phosphate
  • Replace with normal nucleotide, ligate

Fig. 33
Eukaryotic BER
Fig. 34
  • DNA polymerase b fills in
  • missing nucleotide
  • Makes mistakes, not proofread
  • APE1 proofreads
  • (AP endonuclease)
  • Repair of 8-oxoG sites is special case of BER
  • After replication, A often is inserted A can be
    removed by specialized adenine DNA glycosylase
  • Before replication, oxoG paired with C the oxoG
    is removed by oxoG DNA glycoslyase (hOGG1)

Nucleotide Excision Repair (NER)
  • NER handles bulky damage that distorts DNA
  • Including Thymine dimers, large adducts
  • Specific endonucleases clip DNA strand on either
    side of lesion, remove single strand,
    resynthesize and rejoin.
  • Xeroderma pigmentosum
  • (XP) people have hereditary
  • increased skin cancer
  • lack NER enzymes

NER in E. coli
Fig. 36
  • Excinuclease (UvrABC) cuts either side of damage
  • Remove 12-13 nt oligonucleotide
  • Pol I fills in using top strand as template
  • DNA ligase seals nick

Eukaryotic NER uses 2 paths
  • GG-NER
  • Complex of XPC and hHR23B initiates repair, binds
  • limited DNA melting
  • XPA and RPA recruited
  • TFIIH joins, helicase expands melted region
  • RPA binds 2 excinucleases (XPF, XPG) cleaves
  • Releases damage 24-32 nt
  • Transcription-Coupled (TC-NER) resembles GG-NER
  • RNA polymerase plays role of XPC in damage
    sensing and initial DNA melting

Human Global Genome NER
  • Complex of XPC and hHR23B initiates repair, binds
  • Limited DNA melting
  • XPA RPA recruited
  • TFIIH joins, helicase expands melted region
  • RPA binds 2 excinucleases (XPF, XPG),
  • Cleaves, releases damage 24-32 nt

Fig. 37
Transcription-Coupled (TC)-NER
  • Resembles GG-NER
  • RNA polymerase plays role of XPC in damage
    sensing and initial DNA melting
  • RNAP stalls

Fig. 37
Double-Strand Break (DSB) Repair in Eukaryotes
  • dsDNA breaks in eukaryotes are very dangerous
  • Broken chromosomes
  • If not repaired, lead to cell death
  • In vertebrates, also leads to cancer
  • Eukaryotes deal with dsDNA breaks in 2 ways
  • Homologous recombination with good chromsome
  • Nonhomologous end-joining (NHEJ) has errors
  • Chromatin remodeling has role in dsDNA break

Model for Nonhomologous End-Joining
  • Ku and DNA-PKcs bind at DNA ends and let ends
    find microhomology
  • 2 DNA-PK complexes phosphorylate each other
  • Catalytic subunit dissociates
  • DNA helicase activity of Ku unwinds DNA ends
  • Extra flaps of DNA removed, gaps filled, ends
  • Inaccurate process, DNA is lost

Fig. 38
Mismatch Repair
  • Recognizes parental DNA by its methylated A in
    GATC sequence (E. coli)
  • Corrects mismatch in progeny strand
  • Eukaryotes use part of repair system unclear
    how distinguish strands at mismatch
  • HNPCC colon cancer- defects in repair of mismatch
    damage cause instability of microsatellite
    regions, many mutations

Fig. 39
Coping with DNA Damage Without Repairing It
  • Direct reversal and excision repair are true
    repair processes - accurate
  • Eliminate defective DNA entirely
  • Cells also copes with damage by skirting it
  • Not true repair mechanism
  • Damage bypass mechanism
  • gives time to repair
  • cell can replicate, fix damage later

Recombination Repair
  • Gapped DNA strand across from damaged strand
    recombines with normal strand in other daughter
    DNA duplex after replication
  • Must occur before segregation
  • Solves gap problem
  • Leaves original damage unrepaired fix later

Fig. 40
Error-Prone Bypass (SOS)
  • Induce SOS response
  • Activates recA protease
  • UmuC/D dimer is DNA pol V
  • Causes DNA to replicate even though damaged
    region not read correctly
  • Errors in newly made DNA, but cell lives
  • Mutants of umu genes die, but do not have

Fig. 41
Recall, UV damage to cell can induce SOS path,
which causes cleavage of lambda repressor, return
of prophage to lytic cycle
Error-Prone Bypass in Humans
  • Humans have relatively error-free bypass system
    that inserts dAMPs across from pyrimidine dimer
  • Specialized DNA polymerases are activated
  • Replicate thymine dimers correctly
  • Uses DNA polymerase ? plus another enzyme to
    replicate a few bases beyond lesion
  • Polymerase is not really error-free
  • If DNA polymerase ? gene is defective, DNA
    polymerase ? and others take over, more errors

Review questions
  • 8. Diagram rolling circle replication of lambda
  • 12, 16. List the different DNA polymerases in E.
    coli and eukaryotes and explain their roles.
  • 24. Compare/ contrast base excision repair and
    nucleotide excision.
  • 33. Diagram recombination repair in E.coli.