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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”
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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.
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Lewin’s Genes X, 2009.
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FIGURE 02: Replicated DNA is seen as a replication bubble flanked
by nonreplicated DNA
Origin Lewin’s Genes X, 2009.
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Robberson & Clayton, 1972. PNAS 69:3810-4 FIGURE 11.5
Lewin’s Genes X, 2009.
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Lewin’s Genes X, 2009.
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Lewin’s Genes X, 2009.
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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.
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Principles of Two Dimensional-Neutral Agarose Gel Electrophoresis (2D-NAGE)
Priit Joers
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Principles of 2D-NAGE Priit Joers
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Principles of 2D-NAGE Priit Joers
go for a Southern blot...
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Principles of 2D-NAGE Priit Joers
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Origin within fragment -bubble arc Priit Joers
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Nicking of DNA – broken bubbles Priit Joers
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Passing replication fork – Y arc Priit Joers
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ssDNA regions – sub-Y arc Priit Joers
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Colliding forks – double Y and X Priit Joers
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Replication Intermediates
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Holt et al., 2000. Cell 100:515-24
Human
143B
Replication Intermediates
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Holt et al., 2000. Cell 100:515-24
Mouse
Replication Intermediates
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Bacteria
Lewin’s Genes X, 2009.
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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
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Replication Fork Trap
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Replication Fork Trap
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Replication Fork Trap
ter sites
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Replication Fork Trap
ter sites
Tus protein
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Replication Fork Trap
Kamada et al., 1996. Nature 383:598-603
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Replication Fork Trap
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Replication Fork Trap
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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
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origin
melting
Lewin’s Genes X, 2009.
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HU origin
melting
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14.2 Initiation: Creating the Replication Forks at the Origin oriC
SSB
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14.2 Initiation: Creating the Replication Forks at the Origin oriC
gyrase
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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
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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.
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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
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SeqA protein
Kaguni, 2006. ARM 60: 351-71.
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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
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Hda
Regulatory Inactivation of DnaA (RIDA)
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Hansen et al., 2007. JMB 367:942-52.
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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
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Helicase + Helicase Loader
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DnaB Structure
Bailey et al., 2007. Science 318:459-63.
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The Prepriming Complex of E. coli
Mott et al., 2008. Cell 135:623-34.
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Transition from Initiation to Elongation
Makowska-Grzyska & Kaguni, 2010. Mol Cell 37:90-101.
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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
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Transition from Initiation to Elongation
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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.
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E. coli pol III core
Subunits
• α – 5’-3’ polymerase activity
• ε – 3’-5’ exonuclease activity
• θ – stimulate ε
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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.
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The Processivity Factor
(Sliding Clamp)
http://biology.jbpub.com/book/genes/animations/g2480.swf
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The Clamp Loader
Jeruzalmi et al, 2001. Cell 106:429-41.
Kelch et al, 2011. Science 334:1675-80.
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The Clamp Loader
Jeruzalmi et al, 2001. Cell 106:429-41.
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The Clamp Loader
Jeruzalmi et al, 2001. Cell 106:429-41.
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E. coli pol III holoenzyme
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Loading the Polymerase
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Loading the Polymerase
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Putting the pieces together:
The E. coli Replisome
McHenry, 2011. COCB 15:587-94.
Leading
strand
Lagging
strand
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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
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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)
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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.
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14.12 The Clamp Controls Association of
Core Enzyme with DNA
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http://www.wehi.edu.au/education/wehitv/molecular_visualisations_of_dna/
E. coli DNA replication
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The E. coli Replisome
Trimeric polymerase?
Reyes-Lamothe et al., 2010. Science 328:498-501.
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Georgescu et al., 2012. NSMB 19:113-6.
The E. coli Replisome
Trimeric polymerase?
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Graham et al., 2017. Cell 169:1201-13.
Coordination of leading and lagging strand syntheses
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Coordination of leading and lagging strand syntheses
Graham et al., 2017. Cell 169:1201-13.
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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.
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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.
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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.
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• 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
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• 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
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FIGURE 13: The eukaryote cell cycle
Eukaryotes
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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
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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.
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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)
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ORC1 ORC2 ORC3
ORC6 ORC5
ORC4
Cdc6
MCM7
MCM2 MCM
3
MCM6 MCM
5
MCM4
MCM7
MCM2 MCM
3
MCM6 MCM
5
MCM4
Cdt1
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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.
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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
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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.
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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
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• 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
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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)
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FIGURE 27: Similar functions are required at all replication forks
Lewin’s Genes X, 2009.
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Eukaryotic Nucleus (Archea)
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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
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Eukaryotic Nucleus (Archea)
Pol α/primase
• RNA stretch of 11 nt + DNA stretch of variable sizes
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Eukaryotic Nucleus (Archea)
Replication Protein A (RPA) – the SSB
E. coli SSB
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Eukaryotic Nucleus (Archea)
Proliferating Cell Nuclear Antigen (PCNA) – the Sliding Clamp
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Eukaryotic Nucleus (Archea)
Replication Factor C (RFC) – the Clamp Loader
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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.
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Eukaryotic Nucleus (Archea)
Primer Removal and Maturation of Okazaki fragments
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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?
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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
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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
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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
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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
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• 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
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• 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