Biologia Genômica 2º Semestre, 2017 Replicação de DNA em ...· Okazaki fragment to the 5′ beginning

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Biologia Genmica

2 Semestre, 2017

Replicao de DNA em Bactrias e no

Ncleo Eucaritico

Prof. Marcos Tlio

mtoliveria@fcav.unesp.br

Faculdade de Cincias Agrrias e Veterinrias de Jaboticabal

Instituto de Biocincias, Letras e Cincias Exatas de S.J.R.P.

Universidade Estadual Paulista Jlio de Mesquita Filho

mailto:mtoliveria@fcav.unesp.br

DNA Molecules

DNA Molecules

DNA Molecules

11.1 Introduction

replicon A unit of the genome in which DNA is

replicated. Each contains an origin for initiation of

replication.

origin A sequence of DNA at which replication is

initiated.

terminus A segment of DNA at which replication ends.

Lewins Genes X, 2009.

Lewins Genes X, 2009.

FIGURE 02: Replicated DNA is seen as a replication bubble flanked

by nonreplicated DNA

Origin Lewins Genes X, 2009.

Robberson & Clayton, 1972. PNAS 69:3810-4 FIGURE 11.5

Lewins Genes X, 2009.

Lewins Genes X, 2009.

Lewins Genes X, 2009.

11.3 Origins Can Be Mapped by

Autoradiography and Electrophoresis

Replication forks create Y-shaped structures that change

the electrophoretic migration of DNA molecules.

FIGURE 07: The

position of the origin and

the number of replicating

forks determine the

shape of a replicating

restriction fragment

Lewins Genes X, 2009.

Principles of Two Dimensional-Neutral Agarose Gel Electrophoresis (2D-NAGE)

Priit Joers

Principles of 2D-NAGE Priit Joers

Principles of 2D-NAGE Priit Joers

go for a Southern blot...

Principles of 2D-NAGE Priit Joers

Origin within fragment -bubble arc Priit Joers

Nicking of DNA broken bubbles Priit Joers

Passing replication fork Y arc Priit Joers

ssDNA regions sub-Y arc Priit Joers

Colliding forks double Y and X Priit Joers

Replication Intermediates

Holt et al., 2000. Cell 100:515-24

Human

143B

Replication Intermediates

Holt et al., 2000. Cell 100:515-24

Mouse

Replication Intermediates

Bacteria

Lewins Genes X, 2009.

11.4 The Bacterial Genome Is (Usually) a

Single Circular Replicon

The two replication

forks usually meet

halfway around the

circle, but there are ter

sites that cause

termination if the

replication forks go too

far.

FIGURE 09: Forks usually meet

before terminating

Replication Fork Trap

Replication Fork Trap

Replication Fork Trap

ter sites

Replication Fork Trap

ter sites

Tus protein

Replication Fork Trap

Kamada et al., 1996. Nature 383:598-603

Replication Fork Trap

Replication Fork Trap

Initial steps at oriC.

Carr K M , Kaguni J M J. Biol. Chem. 2001;276:44919-44925

2001 by American Society for Biochemistry and Molecular Biology

origin

melting

Lewins Genes X, 2009.

HU origin melting

14.2 Initiation: Creating the Replication Forks at the Origin oriC

SSB

14.2 Initiation: Creating the Replication Forks at the Origin oriC

gyrase

Initial steps at oriC.

Carr K M , Kaguni J M J. Biol. Chem. 2001;276:44919-44925

2001 by American Society for Biochemistry and Molecular Biology

11.5 Methylation of the Bacterial Origin

Regulates Initiation

oriC contains binding sites for DnaA dnaA-boxes.

oriC also contains eleven GATC/CTAG repeats that are

methylated on adenine on both strands.

11.5 Methylation of the Bacterial Origin

Regulates Initiation

Replication generates

hemimethylated DNA,

which cannot initiate

replication.

There is a 13-minute

delay before the

GATC/CTAG repeats

are remethylated.

FIGURE 11: Only fully methylated origins can initiate replication

SeqA protein

SeqA protein

Kaguni, 2006. ARM 60: 351-71.

DnaA (ATP)

dnaA

dnaA

Initial steps at oriC.

Carr K M , Kaguni J M J. Biol. Chem. 2001;276:44919-44925

2001 by American Society for Biochemistry and Molecular Biology

Hda

Regulatory Inactivation of DnaA (RIDA)

Hansen et al., 2007. JMB 367:942-52.

Regulation of Initiation of DNA Replication in

Bacteria (E. coli) All About DnaA

Hemimethylation of oriC

Sequestration of oriC by SeqA.

Hemimethylation of dnaA gene promoter

Hydrolysis of ATP by DnaA + Hda

Titration of DnaA by datA locus

Helicase + Helicase Loader

DnaB Structure

Bailey et al., 2007. Science 318:459-63.

The Prepriming Complex of E. coli

Mott et al., 2008. Cell 135:623-34.

Transition from Initiation to Elongation

Makowska-Grzyska & Kaguni, 2010. Mol Cell 37:90-101.

Transition from Initiation to Elongation

Bailey et al., 2007. Science 318:459-63.

Corn et al., 2008. NSMB 15:163-9.

DnaB + DnaG (model)

DnaG primase

Transition from Initiation to Elongation

E. coli pol III holoenzyme

Subunits

Catalytic core: (pol activity), (exo

activity), (?)

Processivity factor: 2 (sliding clamp)

Clamp Loader (DnaX/ complex): ,

2, , , , .

Lewins Genes X, 2009.

E. coli pol III core

Subunits

5-3 polymerase activity

3-5 exonuclease activity

stimulate

14.5 DNA

Polymerases Control

the Fidelity of

Replication DNA polymerases often

have a 35 exonuclease

activity that is used to

excise incorrectly paired

bases.

The fidelity of replication is

improved by proofreading

by a factor of ~100.

Lewins Genes X, 2009.

The Processivity Factor

(Sliding Clamp)

http://biology.jbpub.com/book/genes/animations/g2480.swf

The Clamp Loader

Jeruzalmi et al, 2001. Cell 106:429-41.

Kelch et al, 2011. Science 334:1675-80.

The Clamp Loader

Jeruzalmi et al, 2001. Cell 106:429-41.

The Clamp Loader

Jeruzalmi et al, 2001. Cell 106:429-41.

E. coli pol III holoenzyme

Loading the Polymerase

Loading the Polymerase

Putting the pieces together:

The E. coli Replisome

McHenry, 2011. COCB 15:587-94.

Leading

strand

Lagging

strand

Putting the pieces together:

The E. coli Replisome

McHenry, 2011. COCB 15:587-94.

Leading

strand

Lagging

strand

links Pol III HE to DnaB/DnaG

Putting the pieces together:

The E. coli Replisome

McHenry, 2011. COCB 15:587-94. link Pol III HE to SSB

SSB + ssDNA

DnaG binds SSB (ssDNA)

14.12 The Clamp

Controls Association of

Core Enzyme with DNA

The helicase DnaB is

responsible for interacting

with the primase DnaG to

initiate each Okazaki

fragment.

FIGURE 21: Each catalytic core of Pol

III synthesizes a daughter strand. DnaB

is responsible for forward movement at

the replication fork

Lewins Genes X, 2009.

14.12 The Clamp Controls Association of

Core Enzyme with DNA

http://www.wehi.edu.au/education/wehitv/molecular_visualisations_of_dna/

E. coli DNA replication

http://www.wehi.edu.au/education/wehitv/molecular_visualisations_of_dna/http://www.wehi.edu.au/education/wehitv/molecular_visualisations_of_dna/

The E. coli Replisome

Trimeric polymerase?

Reyes-Lamothe et al., 2010. Science 328:498-501.

Georgescu et al., 2012. NSMB 19:113-6.

The E. coli Replisome

Trimeric polymerase?

Graham et al., 2017. Cell 169:1201-13.

Coordination of leading and lagging strand syntheses

Coordination of leading and lagging strand syntheses

Graham et al., 2017. Cell 169:1201-13.

14.13 Okazaki

Fragments Are

Linked by Ligase

Each Okazaki fragment

starts with a primer and

stops before the next

fragment.

RNase H + DNA

polymerase I removes

the primer and replaces

it with DNA.

Lewins Genes X, 2009.

14.13 Okazaki Fragments Are Linked by Ligase

DNA ligase makes the bond that

connects the 3 end of one

Okazaki fragment to the 5

beginning of the next fragment.

FIGURE 25: DNA ligase seals nicks

between adjacent nucleotides by

employing an enzyme-AMP intermediate

Lewins Genes X, 2009.

E. coli DNA replication Summary

DnaA melts oriC and recruits DnaB helicase/DnaC

helicase loader.

DnaB helicase recruits DnaG primase. Priming

releases DnaC from prepriming complex.

DnaB helicase keeps interacting with DnaG primase

transiently throughout lagging-strand synthesis.

DnaX clamp loader loads 2 clamp on primer-template

(via interactions with subunit). Pol III core ( subunit)

interacts with 2 clamp and primer-template.

Two (Three!) Pol III cor