Biologia Genômica 2º Semestre, 2017 Replicação de DNA em ...€¦ · 11.1 Introduction • replicon – A unit of the genome in which DNA is replicated. Each contains an origin

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”

mailto:[email protected]

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.


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




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


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

Priit Joers

Principles of 2D-NAGE Priit Joers


go for a Southern blot...


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


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

Mouse


Bacteria


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



ter sites


ter sites

Tus protein


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



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


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




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




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.


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

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

DnaB + DnaG (model)

DnaG primase


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.


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.


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


The Clamp Loader


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



McHenry, 2011. COCB 15:587-94.

Leading

strand

Lagging

strand

τ links Pol III HE to DnaB/DnaG



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


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




Trimeric polymerase?

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

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


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.


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


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!)


• 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.


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


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


Pol α/primase

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


Replication Protein A (RPA) – the SSB

E. coli SSB


Proliferating Cell Nuclear Antigen (PCNA) – the Sliding Clamp


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.


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

Documents

Biologia Genômica 2º Semestre, 2017 Replicação de DNA em ...€¦ · 11.1 Introduction • replicon – A unit of the genome in which DNA is replicated. Each contains an origin