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REPLICATION


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REPLICATION. Chapter 7. The Problem. DNA is maintained in a compressed, supercoiled state. BUT, basis of replication is the formation of strands based on specific bases pairing with their complementary bases.  Before DNA can be replicated it must be made accessible, i.e., it must be unwound.

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REPLICATION

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Replication

REPLICATION

Chapter 7


The problem

The Problem

  • DNA is maintained in a compressed, supercoiled state.

  • BUT, basis of replication is the formation of strands based on specific bases pairing with their complementary bases.

  •  Before DNA can be replicated it must be made accessible, i.e., it must be unwound


Models of replication

Models of Replication

THREE HYPOTHESES FOR DNA REPLICATION


Replication

MODELS OF DNA REPLICATION

(a) Hypothesis 1:

(b) Hypothesis 2:

(c) Hypothesis 3:

Semi-conservative replication

Conservative replication

Dispersive replication

Intermediate molecule


Replication

PREDICTED DENSITIES OF

NEWLY REPLICATED DNA

MOLECULES ACCORDING

TO THE THREE HYPOTHESES

ABOUT DNA REPLICATION


Replication

Meselson and Stahl

Conclusion: Semi-conservative replication of DNA


Replication

Replication as a process

  • Double-stranded DNA unwinds.

The junction of the unwound

molecules is a replication fork.

A new strand is formed by pairing

complementary bases with the

old strand.

Two molecules are made.

Each has one new and one old

DNA strand.


Extending the chain

Extending the Chain

  • dNTPs are added individually

  • Sequence determined by pairing with template strand

  • DNA has only one phosphate between bases, so why use dNTPs?


Extending the chain1

Extending the Chain


Dna synthesis

DNA Synthesis

3’-OH nucleophilic attack on alpha phosphate of incoming dNTP

removal and splitting of pyrophosphate by inorganic pyrophosphatase

2 phosphates


Replication

Chain Elongation in the5’  3’ direction


Semi discontinuous replication

Semi-discontinuous Replication

  • All known DNA pols work in a 5’>>3’ direction

  • Solution?

    • Okazaki fragments


Okazaki experiment

Okazaki Experiment


Replication

Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous


Features of dna replication

Features of DNA Replication

  • DNA replication is semiconservative

    • Each strand of template DNA is being copied.

  • DNA replication is semidiscontinuous

    • The leading strand copies continuously

    • The lagging strand copies in segments (Okazaki fragments) which must be joined

  • DNA replication is bidirectional

    • Bidirectional replication involves two replication forks, which move in opposite directions


Dna replication prokaryotes

DNA Replication-Prokaryotes

  • DNA replication is semiconservative. the helix must be unwound.

  • Most naturally occurring DNA is slightly negatively supercoiled.

  • Torsional strain must be released

  • Replication induces positive supercoiling

  • Torsional strain must be released, again.

  • SOLUTION: Topoisomerases


The problem of overwinding

The Problem of Overwinding


Topoisomerase type i

Topoisomerase Type I

  • Precedes replicating DNA

  • Mechanism

    • Makes a cut in one strand, passes other strand through it. Seals gap.

    • Result: induces positive supercoiling as strands are separated, allowing replication machinery to proceed.


Helicase

Helicase

  • Operates in replication fork

  • Separates strands to allow DNA Pol to function on single strands.

    Translocate along single strain in 5’->3’ or 3’-> 5’ direction by hydrolyzing ATP


Gyrase a type ii topoisomerase

Gyrase--A Type II Topoisomerase

  • Introduces negative supercoils

  • Cuts both strands

  • Section located away from actual cut is then passed through cut site.


Initiation of replication

Initiation of Replication

  • Replication initiated at specific sites: Origin of Replication (ori)

  • Two Types of initiation:

    • De novo –Synthesis initiated with RNA primers. Most common.

    • Covalent extension—synthesis of new strand as an extension of an old strand (“Rolling Circle”)


De novo initiation

De novo Initiation

  • Binding to Ori C by DnaA protein

  • Opens Strands

  • Replication proceeds bidirectionally


Unwinding the dna by helicase dnab protein

Unwinding the DNA by Helicase (DnaB protein)

  • Uses ATP to separate the DNA strands

  • At least 4 helicases have been identified in E. coli.

  • How was DnaB identified as the helicase necessary for replication?

  • NOTE: Mutation in such an essential gene would be lethal.

  • Solution?

    • Conditional mutants


Liebowitz experiment

Liebowitz Experiment

What would you expect if the substrates are separated by electrophoresis after treatment with a helicase?


Liebowitz assay results

Liebowitz Assay--Results

  • What do these results indicate?

  • ALTHOUGH PRIMASE (DnaG) AND SINGLE- STRAND BINDING PROTEIN (SSB) BOTH STIMULATE DNA HELICASE (DnaB), NEITHER HAVE HELICASE ACTIVITY OF THEIR OWN


Single stranded dna binding proteins ssb

Single Stranded DNA Binding Proteins (SSB)

  • Maintain strand separation once helicase separates strands

  • Not only separate and protect ssDNA, also stimulates binding by DNA pol (too much SSB inhibits DNA synthesis)

  • Strand growth proceeds 5’>>3’


Replication the overview

Replication: The Overview

  • Requirements:

    • Deoxyribonucleotides

    • DNA template

    • DNA Polymerase

      • 5 DNA pols in E. coli

      • 5 DNA pols in mammals

    • Primer

  • Proofreading


The dna polymerase family

The DNA Polymerase Family

A total of 5 different DNAPs have been reported in E. coli

  • DNAP I: functions in repair and replication

  • DNAP II: functions in DNA repair (proven in 1999)

  • DNAP III: principal DNA replication enzyme

  • DNAP IV: functions in DNA repair (discovered in 1999)

  • DNAP V: functions in DNA repair (discovered in 1999)

    To date, a total of 14 different DNA polymerases have been reported in eukaryotes


Dna pol i

DNA pol I

  • First DNA pol discovered.

  • Proteolysis yields 2 chains

    • Larger Chain (Klenow Fragment) 68 kd

      • C-terminal 2/3rd. 5’>>3’ polymerizing activity

      • N-terminal 1/3rd. 3’>>5’ exonuclease activity

    • Smaller chain: 5’>>3 exonucleolytic activity

      • nt removal 5’>>3’

      • Can remove >1 nt

      • Can remove deoxyribos or ribos


Dna pol i1

DNA pol I

  • First DNA pol discovered.

  • Proteolysis yields 2 chains

    • Larger Chain (Klenow Fragment) 68 kd

      • C-terminal 2/3rd. 5’>>3’ polymerizing activity

      • N-terminal 1/3rd. 3’>>5’ exonuclease activity

    • Smaller chain: 5’>>3 exonucleolytic activity

      • nt removal 5’>>3’

      • Can remove >1 nt

      • Can remove deoxyribos or ribos


Replication

The structure of the

Klenow fragment of

DNAP I from E. coli


Nick translation

Nick Translation

  • Requires 5’-3’ activity of DNA pol I

  • Steps

  • At a nick (free 3’ OH) in the DNA the DNA pol I binds and digests nucleotides in a 5’-3’ direction

  • The DNA polymerase activity synthesizes a new DNA strand

  • A nick remains as the DNA pol I dissociates from the ds DNA.

  • The nick is closed via DNA ligase

Source: Lehninger pg. 940


Nick translation 2

Nick Translation 2

  • 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation"

This activity is critical in primer removal


Dna polymerase i is great but

DNA Polymerase I is great, but….

In 1969 John Cairns and Paula deLucia

-isolated a mutant bacterial strain with only 1% DNAP I activity (polA)

- mutant was super sensitive to UV radiation

- but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA

Conclusion:

  • DNA pol I is NOT the principal replication enzyme in E. coli


Other clues

Other clues….

- DNAP I is too slow (600 dNTPs added/minute)

- DNAP I is only moderately processive

(processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template)

Conclusion:

  • There must be additional DNA polymerases.

  • Biochemists purified them from the polA mutant


Dna polymerase iii

DNA Polymerase III

The major replicative polymerase in E. coli

  • ~ 1,000 dNTPs added/sec

  • It’s highly processive: >500,000 dNTPs added before dissociating

  • Accuracy:

    • 1 error in 107 dNTPs added,

    • with proofreading final error rate of 1 in 1010 overall.


Dna polymerase iii holoenzyme replicase

The 10 subunits of E. coli DNA polymerase III

5’ to 3’ polymerizing activity

3’ to 5’ exonuclease activity

a and e assembly (scaffold)

Assembly of holoenzyme on DNA

Sliding clamp = processivity factor

Clamp-loading complex

Clamp-loading complex

Clamp-loading complex

Clamp-loading complex

Clamp-loading complex

a

e

q

t

b

g

d

d’

c

y

Core

enzyme

Holoenzyme

DNA Polymerase III Holoenzyme (Replicase)

Subunit

Function


Activities of dna pol iii

Activities of DNA Pol III

  • ~900 kd

  • Synthesizes both leading and lagging strand

  • Can only extend from a primer (either RNA or DNA), not initiate

  • 5’>>3’ polymerizing activity

  • 3’>>5’ exonuclease activity

  • NO 5’>>3’ exonuclease activity


Replication

The 5’ to 3’ DNA polymerizing activity

Subsequent

hydrolysis of

PPi drives the

reaction forward

Nucleotides are added at the 3'-end of the strand


Leading and lagging strands

Leading and Lagging Strands

  • REMEMBER: DNA polymerases require a primer.

  • Most living things use an RNA primer

  • Leading strand (continuous): primer made by RNA polymerase

  • Lagging strand (discontinuous): Primer made by Primase

    • Priming occurs near replication fork, need to unwind helix. SOLUTION: Helicase

    • Primosome= Primase + Helicase


The replisome

The Replisome

  • DNA pol III extends on both the leading and lagging strand

  • Growth stops when Pol III encounters an RNA primer (no 5’>>3’ exonuclease activity)

  • Pol I then extends the chain while removing the primer (5’>>3’)

  • Stops when nick is sealed by ligase


Ligase

Ligase

  • Uses NAD+ or ATP for coupled reaction

  • 3-step reaction:

    • AMP is transferred to Lysine residue on enzyme

    • AMP transferred to open 5’ phosphate via temporary pyrophosphate (i.e., activation of the phosphate in the nick)

    • AMP released, phosphodiester linkage made

  • NADNMN + AMP

  • ATP ADP + PPi


Dna replication model

DNA Replication Model

  • Relaxation of supercoiled DNA.

  • Denaturation and untwisting of the double helix.

  • Stabilization of the ssDNA in the replication fork by SSBs.

  • Initiation of new DNA strands.

  • Elongation of the new DNA strands.

  • Joining of the Okazaki fragments on the lagging strand.


Termination of replication

Termination of Replication

  • Occurs @ specific site opposite ori c

  • ~350 kb

  • Flanked by 6 nearly identical non-palindromic*, 23 bp terminator (ter) sites

  • * Significance?

Tus Protein-arrests replication fork motion


Fidelity of replication

FIDELITY OF REPLICATION

  • Expect 1/103-4, get 1/108-10.

  • Factors

    • 3’5’ exonuclease activity in DNA pols

    • Use of “tagged” primers to initiate synthesis

    • Battery of repair enzymes

    • Cells maintain balanced levels of dNTPs


Why okazaki frags

Why Okazaki Frags?

  • Or, why not 3’5’ synthesis?

  • Possibly due to problems with proofreading.

  • PROBLEM:

    • Imagine a misincorporation with a 3’5’ polymerase

    • How is it removed?

    • How is the chain extended?

    • Is there a problem after removing a mismatch?


Covalent extension methods

Covalent Extension Methods

  • Often called “Rolling circle”

  • Common in bacteriophages

  • NOTE: de novo initiation of circular DNA results in theta structures, sometimes callled “theta replication”


Rolling circle i

Rolling Circle I

  • Few rounds of theta-replication

  • Nick outer strand

  • Extend 3’ end of outer strand, displacing original

  • Synthesis of complementary strand using displaced strand as template

  • Concatamers cut by RE’s, sealed

  • Result several copies of circular dsDNA


Rolling circle i1

Rolling Circle I

  • “Template “rolls”, extrudes leading strand

  • Okazaki frags made on leading strand as it emerges.


Rolling circle i2

Rolling Circle I

NOTE: can get single unit genomes or multimeric copies


Rolling circle ii

Rolling Circle II

  • EX ΦX174

  • Circular ssDNA chromosome

  • Copy + strand using E. coli replication proteins to make ds circle (theta replication)

  • Protein A (phage) cuts + strand

  • Rolling circle replication

  • Protein A cuts at unit length and circularizes (ligates) released ss chromosome

  • Replication continues


Reverse transcription

Reverse Transcription

  • DNA replication in retroviruses

  • RNA Dependent DNA polymerase

  • Process:

    • Retroviral RNA acts as template

      • Primer—Segment of host cell t-RNA

      • Result: DNA RNA hybrid

    • RNA strand degraded by RNAse H

    • DNA strand serves as template.

      • Also catalyzed by RT

      • Result:dsDNA

    • New DNA integrates into host genome


Cdna library

cDNA Library

  • Made from mRNA

  • Steps

    • 1st strand

    • RNAse H

    • 2nd strand

    • Tailing

    • Insertion

    • Transform


Eukaryotic dna replication

Eukaryotic DNA Replication

  • Much larger genomes with slower polymerase

  • Solution

    • Multiple initiation sites

    • More molecules of polymerase

    • EX: DNA pol present in ~2-5 X105 copies/cell

  • Histones an issue

  • Still many questions


Completing the ends of non circular dna

Completing the Ends of Non-circular DNA

  • THE PROBLEM?

  • Solutions

    • Phage T-7

    • Eukaryotes


Phage solution to problem

Phage Solution to Problem

  • Phage DNA is linear

  • Ends have repetitive complementary sequences

  • After removal of 5’end RNA primer, are left with a 3’ overhang

  • Overhangs form H-bonds with complementary overhangs, gaps filled in ligated. RESULT: concatamers

  • RE cuts concatamer into unit length genomes with 5’ overhangs

  • DNA pol extends 3’ ends, resulting in complete unit length genomes.


Eukaryotes

Eukaryotes

  • Solution: Telomeres

  • At ends of chromosomesare non-coding regions, >1000 tandem repeats of GC rich sequence.

  • Telomeric DNA synthesized and maintained by Telomerase

    • Adds tandem repeats of TTGGG

    • Is a ribonucleoprotein, uses internal ribonucleotide sequences as a template


Telomeres

Telomeres

  • Elongation

  • Translocation

  • Elongation

  • New primer synthesis

  • Dna Replication

  • Primer removal

  • Repeat