Transcript

Biologia Genômica

2º Semestre, 2017

Replicação de DNA em Bactérias e no

Núcleo Eucariótico

Prof. Marcos Túlio

[email protected]

Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal

Instituto de Biociências, Letras e Ciências Exatas de S.J.R.P.

Universidade Estadual Paulista “Júlio de Mesquita Filho”

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.

Lewin’s Genes X, 2009.

Lewin’s Genes X, 2009.

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

by nonreplicated DNA

Origin Lewin’s Genes X, 2009.

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

Lewin’s Genes X, 2009.

Lewin’s Genes X, 2009.

Lewin’s 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

Lewin’s 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

Lewin’s 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

Lewin’s 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: α (pol activity), ε (exo

activity), θ (?)

• Processivity factor: β2 (sliding clamp)

• Clamp Loader (DnaX/γ complex): γ,

τ2, δ, δ’, χ, ψ.

Lewin’s Genes X, 2009.

E. coli pol III core

Subunits

• α – 5’-3’ polymerase activity

• ε – 3’-5’ exonuclease activity

• θ – stimulate ε

14.5 DNA

Polymerases Control

the Fidelity of

Replication • DNA polymerases often

have a 3′–5′ exonuclease

activity that is used to

excise incorrectly paired

bases.

• The fidelity of replication is

improved by proofreading

by a factor of ~100.

Lewin’s 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

τ links Pol III HE to DnaB/DnaG

Putting the pieces together:

The E. coli Replisome

McHenry, 2011. COCB 15:587-94. χψ 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

Lewin’s 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

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.

Lewin’s 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

Lewin’s 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 β2 clamp on primer-template

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

interacts with β2 clamp and primer-template.

• Two (Three!) Pol III cores are kept together in the

replisome through the τ subunits of the DnaX clamp

loader.

• In the lagging strand, DnaX clamp loader is constantly

loading β2 clamps onto new primer-templates; it also

promotes Pol III core hopping from the “old” Okazaki

fragment to the “new” primer.

• The τ subunits of DnaX clamp loader are also

important for interacting with DnaB helicase (τ is the

guy!)

E. coli DNA replication – Summary

• The χψ subunits of DnaX clamp loader (τ attaches

them to the ring) interact with SSB transiently, which

interact with DnaG primase transiently.

• RNase H, DNA pol I and DNA ligase are responsible

for the maturation of the Okazaki fragments.

E. coli DNA replication – Summary

FIGURE 13: The eukaryote cell cycle

Eukaryotes

11.7 Each Eukaryotic Chromosome

Contains Many Replicons

• Eukaryotic replicons are

40 to 100 kb in length.

• Individual replicons are

activated at

characteristic times

during S phase.

• Regional activation

patterns suggest that

replicons near one

another are activated at

the same time.

FIGURE 14: A eukaryotic chromosome contains multiple origins of

replication that ultimately merge during replication

FIGURE 15: Replication forks are organized into foci in the nucleus

Photos courtesy of Anthony D. Mills and Ron Laskey, Hutchinson/MRC

Research Center, University of Cambridge.

11.8 Replication Origins Can Be Isolated in

Yeast

• Origins in S. cerevisiae

are short A-T

sequences that have

an essential 11 bp

sequence.

• The ORC is a complex

of six proteins that

binds to an ARS.

FIGURE 16: An ARS extends

for ~50 bp and includes a

consensus sequence (A) and

additional elements (B1–B3)

ORC1 ORC2 ORC3

ORC6 ORC5

ORC4

Cdc6

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

Cdt1

11.9 Licensing Factor Controls Eukaryotic

Rereplication

• Licensing factor is necessary for initiation of replication

at each origin.

• Licensing factor is present in the nucleus prior to

replication, but is removed, inactivated, or destroyed by

replication.

11.9 Licensing Factor

Controls Eukaryotic

Rereplication

• Initiation of another

replication cycle becomes

possible only after

licensing factor reenters

the nucleus after mitosis.

FIGURE 18: Licensing factor in the

nucleus is inactivated after replication

ORC1 ORC2 ORC3

ORC6 ORC5

ORC4

Cdc6

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

Cdt1

• The ORC is a protein complex that is associated with

yeast origins throughout the cell cycle.

• Cdc6 protein is an unstable protein that is synthesized

only in G1.

• Cdc6 binds to ORC and allows MCM proteins to bind.

• Cdt1 facilitates MCM loading on origins.

11.10 Licensing Factor Consists of MCM

Proteins

• When replication is initiated, Cdc6, Cdt1, and MCM

proteins are displaced. The degradation of Cdc6

prevents reinitiation.

ORC1 ORC2 ORC3

ORC6 ORC5

ORC4

Cdc6

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

Cdt1

• Some MCM proteins are in

the nucleus throughout the

cell cycle, but others may

enter only after mitosis.

FIGURE 19: Proteins at the origin

control susceptibility to initiation

Regulation of Initiation of DNA Replication in

Eukaryotes (yeast)

• ORC recognizes the origin

• Cdc6 is rapidly degraded

• Some MCM proteins are licensing factors (only enter the

nucleus when the envelope is disrupted during mitosis)

FIGURE 27: Similar functions are required at all replication forks

Lewin’s Genes X, 2009.

Eukaryotic Nucleus (Archea)

Eukaryotic Nucleus (Archea)

The MCM2-7 helicase

ORC1 ORC2 ORC3

ORC6 ORC5

ORC4

Cdc6

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

MCM7

MCM2 MCM

3

MCM6 MCM

5

MCM4

Cdt1

Eukaryotic Nucleus (Archea)

Pol α/primase

• RNA stretch of 11 nt + DNA stretch of variable sizes

Eukaryotic Nucleus (Archea)

Replication Protein A (RPA) – the SSB

E. coli SSB

Eukaryotic Nucleus (Archea)

Proliferating Cell Nuclear Antigen (PCNA) – the Sliding Clamp

Eukaryotic Nucleus (Archea)

Replication Factor C (RFC) – the Clamp Loader

14.14 Separate Eukaryotic DNA

Polymerases Undertake Initiation and

Elongation

• DNA polymerase ε elongates the leading strand and a

second DNA polymerase δ elongates the lagging strand.

Eukaryotic Nucleus (Archea)

Primer Removal and Maturation of Okazaki fragments

12.2 The Ends of Linear DNA Are a

Problem for Replication

• Special arrangements must be made to replicate the

DNA strand with a 5′ end.

FIGURE 01: Replication could run off the 3’ end of a newly synthesized

linear strand, but could it initiate at a 5’ end?

9.16 Telomeres Have Simple Repeating

Sequences

• The telomere is required for the stability of the

chromosome end.

• A telomere consists of a simple repeat where a C+A-rich

strand has the sequence C>1(A/T)1–4.

FIGURE 27: A typical telomere has a simple repeating structure with a G-T-

rich strand that extends beyond the C-A-rich strand

9.17 Telomeres Seal the Chromosome Ends

and Function in Meiotic Chromosome Pairing

• The protein TRF2 catalyzes a reaction in which the 3′

repeating unit of the G+T-rich strand forms a loop by

displacing its homolog in an upstream

region of the telomere.

Photo courtesy of Jack Griffith, University of North Carolina at Chapel Hill.

FIGURE 29a: A loop

forms at the end of

chromosomal DNA

9.18 Telomeres Are

Synthesized by a

Ribonucleoprotein Enzyme

FIGURE 32: Telomerase

positions itself by base

pairing between the RNA

template and the protruding

single-stranded DNA primer

9.19 Telomeres Are Essential for Survival

• Telomerase is expressed in

actively dividing cells and is

not expressed in quiescent

cells.

• Loss of telomeres results in

senescence.

• Escape from senescence

can occur if telomerase is

reactivated, or via unequal

homologous recombination

to restore telomeres.

FIGURE 33: Mutation in telomerase causes

telomeres to shorten in each cell division

• Coordination of leading- and lagging-strand synthesis

in the eukaryotic nucleus is obscure.

• Primers are synthesized by the heterotetrameric Pol

α/primase. They are ~half RNA, ~half DNA.

• Although no sequence homology is found among

nuclear, bacterial and T4 sliding clamps and clamp

loaders, their general structure is very similar (donut-

shape, 3/6fold symmetry).

Systems other than E. coli DNA replication –

Summary

• The heterotrimeric nuclear RPA has no homology with

the homotetrameric bacterial SSB, despite possessing

similar structural folding domains for binding ssDNA.

• Two distinct polymerases (ε and δ) are required to

leading and lagging strand synthesis, respectively, in

the nucleus.

• Okazaki fragments maturation is accomplished by a

complex with PCNA, Pol δ/β, Fen1 and DNA ligase I.

• A specialized polymerase (telomerase) is responsible

for replication of the chromosomal ends.

Systems other than E. coli DNA replication –

Summary


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