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

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


Models of Replication

THREE HYPOTHESES FOR DNA REPLICATION


MODELS OF DNA REPLICATION

(a) Hypothesis 1:

(b) Hypothesis 2:

(c) Hypothesis 3:

Semi-conservative replication

Conservative replication

Dispersive replication

Intermediate molecule


PREDICTED DENSITIES OF

NEWLY REPLICATED DNA

MOLECULES ACCORDING

TO THE THREE HYPOTHESES

ABOUT DNA REPLICATION


Meselson and Stahl

Conclusion: Semi-conservative replication of DNA


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

  • dNTPs are added individually

  • Sequence determined by pairing with template strand

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


Extending the Chain


DNA Synthesis

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

removal and splitting of pyrophosphate by inorganic pyrophosphatase

2 phosphates


Chain Elongation in the5’  3’ direction


Semi-discontinuous Replication

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

  • Solution?

    • Okazaki fragments


Okazaki Experiment


Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous


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 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


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

  • 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

  • Introduces negative supercoils

  • Cuts both strands

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


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

  • Binding to Ori C by DnaA protein

  • Opens Strands

  • Replication proceeds bidirectionally


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

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


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)

  • 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

  • Requirements:

    • Deoxyribonucleotides

    • DNA template

    • DNA Polymerase

      • 5 DNA pols in E. coli

      • 5 DNA pols in mammals

    • Primer

  • Proofreading


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

  • 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 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


The structure of the

Klenow fragment of

DNAP I from E. coli


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

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

This activity is critical in primer removal


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….

- 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

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.


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

  • ~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


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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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?

  • 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

  • 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

  • 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 I

  • “Template “rolls”, extrudes leading strand

  • Okazaki frags made on leading strand as it emerges.


Rolling Circle I

NOTE: can get single unit genomes or multimeric copies


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

  • 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

  • Made from mRNA

  • Steps

    • 1st strand

    • RNAse H

    • 2nd strand

    • Tailing

    • Insertion

    • Transform


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

  • THE PROBLEM?

  • Solutions

    • Phage T-7

    • Eukaryotes


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

  • 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

  • Elongation

  • Translocation

  • Elongation

  • New primer synthesis

  • Dna Replication

  • Primer removal

  • Repeat


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