genetics lec - chap 10 - replication

8/8/2019 Genetics Lec - Chap 10 - Replication

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DNA ReplicationDNA Replicationand Synthesisand Synthesis

Fundamental GeneticsLecture 10

John Donnie A. Ramos, Ph.D.Dept. of Biological Sciences

College of ScienceUniversity of Santo Tomas

The Flow of Biological InformationThe Flow of Biological Information

DNA

RNA

Protein

Replication

Transcription

Translation

Modes of DNA ReplicationModes of DNA Replication Semiconservative ReplicationSemiconservative Replication

Semiconservative Replication in ProkaryotesSemiconservative 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 cesiumchloride compared to 14N

Semiconservative Replication in ProkaryotesSemiconservative Replication in Prokaryotes

Expected results of the Messelson-Stahl experiment



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Semiconservative Replication in EukaryotesSemiconservative Replication in Eukaryotes

J. Herbert Taylor, PhilipWoods, and Walter Hughes

(1957)

Used root tip cells from Vicia faba (broad bean)

Monitored replication using3H-Thymidine to label DNA

Used autoradiography todetermine the incorporationof 3H-Thymidine

Arrested cells at metaphaseusing colchicine

Replication ofReplication of E. coli E. coli PlasmidPlasmid

Shown by John Cairns (1981) usingradioisotopes 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 replicationtermination

DNA Synthesis in MicroorganismsDNA Synthesis in Microorganisms

DNA polymerase I (928aa) – catalyses the

synthesis of DNA in vitro

(A. Kornberg, 1957)

Requirements:

Deoxyribonucleosidetriphosphates, dNTPs(dATP, dCTP, dGTP, dTTP)

DNA template

Primer

Chain ElongationChain 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)

DNA PolymerasesDNA 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 externaldamage

DNA Pol III – main enzymeinvolved in DNA synthesis

a holoenzyme (>600,000 Da) – forms replisome whenattached to a replication fork.

Replication in ProkaryotesReplication in Prokaryotes

1. Unwinding of DNA helix

2. Initiation of DNA synthesis

3. DNA synthesis proper (elongation)

4. Sealing gaps

5. Proofreading and error correction



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Unwinding of DNA Helix 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 SynthesisInitiation of DNA Synthesis

Synthesis of RNA primer – 5 to 15 RNA bases complementary tothe DNA template

Catalysed by primase (an RNA polymerase)

Pimase does not require free 3’ end to initiate synthesis (not unlikeDNA polymerase III)

Function of primase will be continued by DNA polymerase III.

DNA Synthesis (Elongation)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) anddiscontinuous in the 5’ to 3’ DNA strand(lagging strand).

Okazaki fragments – short DNA fragments produced in the laggingstrand

Concurrent synthesis of leading andlagging strands occur by using DNA poldimer and by a looping mechanism for

the lagging strand

Sealing of Gaps, ProofreadingSealing of Gaps, Proofreading

and Error Correctionand Error Correction

DNA polymerase I removes all RNA bases produced

by primase (creates gaps in the lagging strand) andreplaces 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)

εεεε subunit of DNA polymerase III is involved in

proofreading.

Assures high fidelity of DNA replication

Mutations Affect ReplicationMutations Affect Replication Replication in EukaryotesReplication in Eukaryotes

Presence of multiple replication origin(faster replication, guaranteesreplication 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.



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Eukaryotic DNA PolymerasesEukaryotic 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 singlesubunit)

Pol ξ - DNA repair

Pol γ - mitochondrial DNA synthesis (encoded by nuclear gene)

Eukaryotes has a high copy number of DNA polymerases (ex. Pol αmay be up to 50K copies)

Eukaryotic DNA PolymerasesEukaryotic DNA Polymerases

Eukaryotic DNA ReplicationEukaryotic DNA Replication

Telomeres – linear ends of

eukaryotic chromosomes

Problem with lagging

strand: no 3’ needed by

DNA polymerase I (afterremoval of RNA primers)

Possible result: chromosomewith shorter lagging strandevery replication step

TelomeraseTelomerase

Enzyme that adds TTGGGGrepeats on the telomeres (first

identified in Tetrahymena)

Prevents shortening of

chromosomes

Forms a “hairpin loop” onchromosome ends using G-G

bonds

Creates a free 3’ on laggingstrand that can be used by

DNA polymerase I to replacedthe removed RNA primer

Telomerase is aribonucleoprotein and contains

RNA sequence (5’ AACCCC 3”-serving as template) – reverse

transcriptase Cleavage of loop after DNA

synthesis

HomologousHomologous

RecombinationRecombination

Exchange of genetic material

Directed by specificenzymes:

Endonuclease – introduces

single strand nicks

Ligase – seals loose ends(nicks)

Rec A protein promotes the

exchange of reciprocal

single-stranded DNA molecules and it enhances

hydrogen bond formation

during strand displacement

Gene ConversionGene Conversion

Exchange of genetic information between non-homologouschromosomes (non-reciprocal genetic exchange)

Type of chromosome mutation (recombination)

First identified in Neurospora (by Mary Mitchell)

Can be repaired but forms recombined genetic material

genetics lec - chap 10 - replication

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