Gene Lecture 10 Replication
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Fundamental Genetics Lecture 10
DNA Replication and Synthesis John Donnie A. Ramos, Ph.D. Dept. of Biological Sciences College of Science University of Santo Tomas
The Flow of Biological Information Replication DNA
Transcription RNA
Translation Protein
1
Modes of DNA Replication
Semiconservative Replication
2
Semiconservative Replication in Prokaryotes Mathew Messelson and Franklin Stahl (1958)
15N
– heavy isotope of N (contains 1 more neutron) compared to 14N
15N
has high sedimentation rate in cesium chloride compared to 14N
Semiconservative Replication in Prokaryotes Expected results of the Messelson-Stahl experiment
3
Semiconservative Replication in Eukaryotes J. Herbert Taylor, Philip Woods, and Walter Hughes (1957) Used root tip cells from Vicia faba (broad bean) Monitored replication using 3H-Thymidine to label DNA Used autoradiography to determine the incorporation of 3H-Thymidine Arrested cells at metaphase using colchicine
Replication of E. coli Plasmid Shown by John Cairns (1981) using radioisotopes and radiography Replication starts in a single OriC – origin of replication (245 bp) Replication is bidirectional Replication fork – unwound DNA helix Replicon – replicated DNA Ter region – region of replication termination
4
DNA Synthesis in Microorganisms DNA polymerase I (928 aa) – catalyses the synthesis of DNA in vitro (A. Kornberg, 1957) Requirements: Deoxyribonucleoside triphosphates, dNTPs (dATP, dCTP, dGTP, dTTP) DNA template Primer
Chain Elongation 5’ to 3’ direction of DNA synthesis (requires 3’ end of the DNA template) Each step incorporates free 3’ OH group for further elongation
DNA replication using DNA polymerase is of high fidelity (highly accurate) With exonuclease activity (proofreading ability)
5
DNA Polymerases All 3 types requires a primer Complex proteins (100,000 Da) Functions of DNA polymerases in vivo DNA Pol I – proofreading; removes primers and fills gaps DNA Pol II - mainly involved in DNA repair from external damage DNA Pol III – main enzyme involved in DNA synthesis a holoenzyme (>600,000 Da) – forms replisome when attached to a replication fork.
Replication in Prokaryotes 1. 2. 3. 4. 5.
Unwinding of DNA helix Initiation of DNA synthesis DNA synthesis proper (elongation) Sealing gaps Proofreading and error correction
6
Unwinding of DNA Helix Takes place in oriC (245 bp) – repeating 9mers and 13mers
Function of helicases (Dna A, B, C) – requires ATP hydrolysis to break hydrogen bonds
Initiated by Dna A – binds to 9mers Binding of Dna B and Dna C to unwound helix
Single-stranded binding proteins (SSBPs) – prevents reannealing of replication bubble.
DNA gyrase (a DNA topoisomerase) – relaxes the supercoiling of DNA helix
Initiation of DNA Synthesis
Synthesis of RNA primer – 5 to 15 RNA bases complementary to the DNA template
Catalysed by primase (an RNA polymerase) Pimase does not require free 3’ end to initiate synthesis (not unlike DNA polymerase III)
Function of primase will be continued by DNA polymerase III.
7
DNA Synthesis (Elongation) Function of DNA polymerase III Requires free 3’ end Direction of elongation: 5’ to 3’ DNA synthesis is continuous in 3’ to 5’
DNA strand (leading strand) and discontinuous in the 5’ to 3’ DNA strand (lagging strand).
Okazaki fragments – short DNA
fragments produced in the lagging strand
Concurrent synthesis of leading and
lagging strands occur by using DNA pol dimer and by a looping mechanism for the lagging strand
Sealing of Gaps, Proofreading and Error Correction DNA polymerase I removes all RNA bases produced
by primase (creates gaps in the lagging strand) and replaces it with DNA bases (U to T).
DNA ligase seals the gaps by forming phosphodiester bonds
Exonuclease proofreading (identification of
mismatched bases) is a function of both DNA polemerase I and III (both with 3’-5’ exonuclease activity)
ε subunit of DNA polymerase III is involved in proofreading.
Assures high fidelity of DNA replication
8
Mutations Affect Replication
Replication in Eukaryotes Presence of multiple replication origin
(faster replication, guarantees replication of a big genome) – 25K replicons in mammalian cells
Autonomously replicating sequences
(ARSs) – origin of replication in yeasts (11 bp)
Origin site is AT rich region Helicase unwinds double stranded DNA
and removes histone proteins from DNA
Histones reassociates while DNA synthesis occurs.
9
Eukaryotic DNA Polymerases Pol α - initiates nuclear DNA synthesis
4 subunits (2 acts primase – produces RNA primers) Acts on both leading and lagging strands 2 other subunits continue elongation step (DNA synthesis) Low processivity (short length of synthesized DNA prior to dissociation)
Pol δ - replaces Pol α (called polymerase switching) High processivity (during elongation) With 3’-5’ exonuclease activity (proofreading)
Pol ε - nuclear DNA synthesis Pol β - DNA repair (the only eukaryotic DNA polymerase with single
subunit) Pol ξ - DNA repair Pol γ - mitochondrial DNA synthesis (encoded by nuclear gene)
Eukaryotes has a high copy number of DNA polymerases (ex. Pol α may be up to 50K copies)
Eukaryotic DNA Replication Telomeres – linear ends of eukaryotic chromosomes
Problem with lagging
strand: no 3’ needed by DNA polymerase I (after removal of RNA primers)
Possible result:
chromosome with shorter lagging strand every replication step
10
Telomerase Enzyme that adds TTGGGG
repeats on the telomeres (first identified in Tetrahymena)
Prevents shortening of chromosomes
Forms a “hairpin loop” on
chromosome ends using G-G bonds
Creates a free 3’ on lagging
strand that can be used by DNA polymerase I to replaced the removed RNA primer
Telomerase is a
ribonucleoprotein and contains RNA sequence (5’ AACCCC 3”serving as template) – reverse transcriptase
Cleavage of loop after DNA synthesis
DNA
Recombination Exchange of genetic material Homologous recombination
Ex. Rec A protein (produces recB, recC and recD genes)
11
Gene Conversion Exchange of genetic information between non-homologous chromosomes
Type of chromosome mutation (recombination) First identified in Neurospora (by Mary Mitchell) Can be repaired but forms recombined genetic material
12
DNA Replication and Synthesis John Donnie A. Ramos, Ph.D. Dept. of Biological Sciences College of Science University of Santo Tomas
The Flow of Biological Information Replication DNA
Transcription RNA
Translation Protein
1
Modes of DNA Replication
Semiconservative Replication
2
Semiconservative Replication in Prokaryotes Mathew Messelson and Franklin Stahl (1958)
15N
– heavy isotope of N (contains 1 more neutron) compared to 14N
15N
has high sedimentation rate in cesium chloride compared to 14N
Semiconservative Replication in Prokaryotes Expected results of the Messelson-Stahl experiment
3
Semiconservative Replication in Eukaryotes J. Herbert Taylor, Philip Woods, and Walter Hughes (1957) Used root tip cells from Vicia faba (broad bean) Monitored replication using 3H-Thymidine to label DNA Used autoradiography to determine the incorporation of 3H-Thymidine Arrested cells at metaphase using colchicine
Replication of E. coli Plasmid Shown by John Cairns (1981) using radioisotopes and radiography Replication starts in a single OriC – origin of replication (245 bp) Replication is bidirectional Replication fork – unwound DNA helix Replicon – replicated DNA Ter region – region of replication termination
4
DNA Synthesis in Microorganisms DNA polymerase I (928 aa) – catalyses the synthesis of DNA in vitro (A. Kornberg, 1957) Requirements: Deoxyribonucleoside triphosphates, dNTPs (dATP, dCTP, dGTP, dTTP) DNA template Primer
Chain Elongation 5’ to 3’ direction of DNA synthesis (requires 3’ end of the DNA template) Each step incorporates free 3’ OH group for further elongation
DNA replication using DNA polymerase is of high fidelity (highly accurate) With exonuclease activity (proofreading ability)
5
DNA Polymerases All 3 types requires a primer Complex proteins (100,000 Da) Functions of DNA polymerases in vivo DNA Pol I – proofreading; removes primers and fills gaps DNA Pol II - mainly involved in DNA repair from external damage DNA Pol III – main enzyme involved in DNA synthesis a holoenzyme (>600,000 Da) – forms replisome when attached to a replication fork.
Replication in Prokaryotes 1. 2. 3. 4. 5.
Unwinding of DNA helix Initiation of DNA synthesis DNA synthesis proper (elongation) Sealing gaps Proofreading and error correction
6
Unwinding of DNA Helix Takes place in oriC (245 bp) – repeating 9mers and 13mers
Function of helicases (Dna A, B, C) – requires ATP hydrolysis to break hydrogen bonds
Initiated by Dna A – binds to 9mers Binding of Dna B and Dna C to unwound helix
Single-stranded binding proteins (SSBPs) – prevents reannealing of replication bubble.
DNA gyrase (a DNA topoisomerase) – relaxes the supercoiling of DNA helix
Initiation of DNA Synthesis
Synthesis of RNA primer – 5 to 15 RNA bases complementary to the DNA template
Catalysed by primase (an RNA polymerase) Pimase does not require free 3’ end to initiate synthesis (not unlike DNA polymerase III)
Function of primase will be continued by DNA polymerase III.
7
DNA Synthesis (Elongation) Function of DNA polymerase III Requires free 3’ end Direction of elongation: 5’ to 3’ DNA synthesis is continuous in 3’ to 5’
DNA strand (leading strand) and discontinuous in the 5’ to 3’ DNA strand (lagging strand).
Okazaki fragments – short DNA
fragments produced in the lagging strand
Concurrent synthesis of leading and
lagging strands occur by using DNA pol dimer and by a looping mechanism for the lagging strand
Sealing of Gaps, Proofreading and Error Correction DNA polymerase I removes all RNA bases produced
by primase (creates gaps in the lagging strand) and replaces it with DNA bases (U to T).
DNA ligase seals the gaps by forming phosphodiester bonds
Exonuclease proofreading (identification of
mismatched bases) is a function of both DNA polemerase I and III (both with 3’-5’ exonuclease activity)
ε subunit of DNA polymerase III is involved in proofreading.
Assures high fidelity of DNA replication
8
Mutations Affect Replication
Replication in Eukaryotes Presence of multiple replication origin
(faster replication, guarantees replication of a big genome) – 25K replicons in mammalian cells
Autonomously replicating sequences
(ARSs) – origin of replication in yeasts (11 bp)
Origin site is AT rich region Helicase unwinds double stranded DNA
and removes histone proteins from DNA
Histones reassociates while DNA synthesis occurs.
9
Eukaryotic DNA Polymerases Pol α - initiates nuclear DNA synthesis
4 subunits (2 acts primase – produces RNA primers) Acts on both leading and lagging strands 2 other subunits continue elongation step (DNA synthesis) Low processivity (short length of synthesized DNA prior to dissociation)
Pol δ - replaces Pol α (called polymerase switching) High processivity (during elongation) With 3’-5’ exonuclease activity (proofreading)
Pol ε - nuclear DNA synthesis Pol β - DNA repair (the only eukaryotic DNA polymerase with single
subunit) Pol ξ - DNA repair Pol γ - mitochondrial DNA synthesis (encoded by nuclear gene)
Eukaryotes has a high copy number of DNA polymerases (ex. Pol α may be up to 50K copies)
Eukaryotic DNA Replication Telomeres – linear ends of eukaryotic chromosomes
Problem with lagging
strand: no 3’ needed by DNA polymerase I (after removal of RNA primers)
Possible result:
chromosome with shorter lagging strand every replication step
10
Telomerase Enzyme that adds TTGGGG
repeats on the telomeres (first identified in Tetrahymena)
Prevents shortening of chromosomes
Forms a “hairpin loop” on
chromosome ends using G-G bonds
Creates a free 3’ on lagging
strand that can be used by DNA polymerase I to replaced the removed RNA primer
Telomerase is a
ribonucleoprotein and contains RNA sequence (5’ AACCCC 3”serving as template) – reverse transcriptase
Cleavage of loop after DNA synthesis
DNA
Recombination Exchange of genetic material Homologous recombination
Ex. Rec A protein (produces recB, recC and recD genes)
11
Gene Conversion Exchange of genetic information between non-homologous chromosomes
Type of chromosome mutation (recombination) First identified in Neurospora (by Mary Mitchell) Can be repaired but forms recombined genetic material
12
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