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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
