Download DNA Replication

Yeast Has Defined Origins
ARS directs autonomous
replication of plasmid DNA
S. cerevisiae ARS contains a
conserved 11 bp ARS consensus
sequence and multiple B elements
The ORC complex binds to the
ARS during most of the cell cycle
The S. pombe origin is larger and
binds ORC by a distinct mechanism
from Bell, Genes Dev. 16, 659 (2002)
Replication Origins in Metazoans
DNA replication initiates from
distinct confined sites
or extended initiation zones
The potential to initiate is modulated
by sequence, supercoiling, transcription,
or epigenetic modifications
Initiation can influence
initiation at an adjacent site
from Aladjem, Nature Rev.Genet. 8, 588 (2007)
Some Features of Eukaryotic Replication Origins
from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)
Certain characteristics are common at metazoan replication origins but are not present at all origins
Different modules contribute to the selection of a given origin
Different Classes of Replication Origins in Metazoans
Only a small subset of origins are
active during a given cell cycle
Constitutive origins are used all
the time and are relatively rare
Flexible origins are used to a
different extent in different
cells and follow the Jesuit Model
“Many are called but few are chosen”
from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)
Inactive or dormant origins are
only used during replication stress
or during certain cellular programs
Chromatin Structure Influences ORC Binding
Chromatin remodelling complexes
can facilitate HAT binding
preRC proteins can be modified by HATs
from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)
Influence of Distal Elements on Initiation
Deletion of DHFR promoter allows
initiation to occur within the gene
Truncation of the DHFR gene confines
initiation to the far end of the locus
Deletion of the b-globin LCR
prevents initiation within the locus
Deletion of the CNS1 sequence
in the Th2 cluster do not
initiate within the IL13 gene
from Aladjem, Nature Rev.Genet. 8, 588 (2007)
The Formation of the preRC
Mcm2-7 is loaded as a double
hexamer by ORC, Cdc6 and Cdt1
Sld3 and Cdc45 bind weakly to Mcm2-7
Mcm2-7 helicase is inactive until S phase
from Labib, Genes Dev. 24, 1208 (2010)
Origins Are Activated at Different Times
preRCs are formed during G1 on origins
Heterochromatic regions replicate
later than euchromatic regions
from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)
The Replicative Helicase
Mcm2-7, Cdc45, and GINS (CMG complex)
form the replicative helicase
from Moyer et al., Proc.Nat.Acad.Sci.USA 103, 10236 (2006)
Assembly of the Replicative Helicase
preRC is formed during G1
by recruitment of Mcm2-7
Phosphorylation of MCM proteins
by DDK recruits GINS and
stabilizes Cdc45 association
from Sheu and Stillman, Mol.Cell 24, 101 (2006)
Helicase Loading and Activation in DNA Replication
DnaA and ORC are structural homologs
Replication competence is
conferred by Mcm2-7 loading
and is prevented by inhibition
of pre-RC proteins
CDKs prevent Mcm2-7 loading and
are required for helicase activation
from Remus and Diffley, Curr.Opin.Cell Biol. 21, 771 (2009)
Activation of Helicase Requires Phosphorylation of Sld2 and Sld3
G1 CDKs allow Dbf4 to accumulate
DDK phosphorylates Mcm2-7
and promotes Cdc45 association
CDK phosphorylates Sld2 and Sld3
and promotes association with Dpb11
from Botchan, Nature 445, 272 (2007)
11-3-2 promotes helicase activation
Division of Labor at the Replication Fork
from Jinks-Robertson and Klein, Nature Rev.Struct.Mol.Biol. 22, 176 (2015)
The Eukaryotic Replication Fork
CMG unwinds parental DNA
CMG recruits Pole
PCNA recruits Pold
Ribonucleotides are incorporated
every 1,000 – 10,000 nucleotides
from Kunkel and Burgers, Nature Struct.Mol.Biol. 21, 649 (2014)
Initiation of Chromosome Replication
DDK phosphorylates Mcm proteins
CDK phosphorylates Sld2 and
Sld3 to interact with Dpb11
GINS and Pol e are recruited
to form the RPC (replisome progression complex)
Activation of the helicase allows priming by Pol a
Pol e extends the leading strand and
Pol d extends each Okazaki fragment
from Labib, Genes Dev. 24, 1208 (2010)
Chromatin Dynamics During DNA Replication
from Ransom et al., Cell 140, 183 (2010)
Nucleosome assembly is coupled to DNA replication
Half of histones on newly replicated DNA are recycled from parental histones
Parental and newly synthesized H2A/H2B dimers and
H3/H4 heterotetramers associate with histone chaperones
Replication Termination
Convergence of replication forks
triggers ubiquitylation of Mcm7
Cdc48 associates with the ubiquitylated
replisome and causes disassembly
from Bell, Science 346, 418 (2014)
Replication Origins are Licensed in Late M and G1
Origins are licensed by Mcm2-7
binding to form part of the pre-RC
Mcm2-7 is displaced as
DNA replication is initiated
Licensing is turned off at late
G1 by CDKs and/or geminin
from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6, 476 (2005)
Control of Licensing Differs in Yeasts and Metazoans
CDK activity prevents licensing in yeast
Geminin activation downregulates
Cdt1 in metazoans
from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6, 476 (2005)
The End Replication Problem
Leading strand is synthesized to
the end of the chromosome
Lagging strand utilizes RNA
primers which are removed
The lagging strand is shortened
at each cell division
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-49
Solutions to the End Replication Problem
3’-terminus is extended using the reverse
transcriptase activity of telomerase
Dipteran insects use retrotransposition with
the 3’-end of the chromosome as a primer
Kluyveromyces lactis uses a rolling circle
mechanism in which the 3’-end is extended
on an extrachromosomal template
Telomerase-deficient yeast use a recombinationdependent replication pathway in which one
telomere uses another telomere as a template
Formation of T-loops using terminal
repeats allow extension of invaded 3’-ends
from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004)
Telomerase Solves the Replication Problem
Telomerase-associated RNA
base pairs to the G overhang
Telomerase catalyzes reverse
transcription to a specific site
Telomerase dissociates and base pairs
to a more 3’-region of the G overhang
Successive reverse transcription,
dissociation, and reannealing
extends the G overhang
The C-strand is filled in using the extended template
from Alberts et al., Molecular Biology of the Cell, 4th ed. Fig 5-43
Telomerase Action is Restricted to a Subset of Ends
Telomere length is regulated by shelterin
Increased levels of shelterin
inhibits telomerase action
from Bertuch and Lundblad, Curr.Opin.Cell Biol. 18, 247 (2006)
Telomeres are Specialized Structures at the Ends of Chromosomes
Telomeres contain multiple
copies of short repeated sequences
and contain a 3’-G-rich overhang
Telomeres are bound by proteins
which protect the telomeric ends
initiate heterochromatin formation
and facilitate progression of the
replication fork
from Gilson and Geli, Nature Rev.Mol.Cell Biol. 8, 825 (2007)
Functions of Telomeres
from Nandakumar and Cech, Nature Rev.Mol.Cell Biol. 14, 69 (2013)
Deprotected telomeres induce checkpoint activation, the DNA damage response and DNA repair
Telomerase is recruited by telomeric proteins and
counteracts telomere shortening during DNA replication
Structure of Human Telomeres
Telomeres consist of numerous short
dsDNA repeats and a 3’-ssDNA overhang
The G-tail is sequestered in the T-loop
Shelterin is a protein complex
that binds to telomeres
TRF1 and TRF2 binds duplex repeat
and recruits other shelterin components
Shelterin inhibits the DNA damage
response and blocks telomerase activity
TRF2 inhibits RNF168 to inhibit NHEJ
from O’Sullivan and Karlseder, Nature Rev.Mol.Cell Biol. 11, 171 (2010)
Maintenance of Chromosome Ends
TPP1 and POT1 recruits telomerase
which extends G-strands
The CST complex binds to the
extended G-strand and suppresses
telomerase access and activity
CST promotes C-strand fill-in by Pola-Primase
from Martinez and Blasco, Trends Biochem.Sci. 40, 504 (2015)
Heterochromatin at Telomeres
Chromosome ends are enriched in
H3K9me3, H4K20me3 and HP1
Subtelomeric DNA is heavily methylated
Telomeric heterochromatin
restricts telomerase access and
suppresses recombination
from Martinez and Blasco, Trends Biochem.Sci. 40, 504 (2015)
G Overhang Generation at Telomeres
TRF2 recruits Apollo at leading telomeres
and initiations overhang generation
POT1 binds to inhibit hyperresection
Exo1 generates elongated overhangs
CST is recruited by POT1 and
recruits Pola-primase to fill in C-strand
from Martinez and Blasco, Trends Biochem.Sci. 40, 504 (2015)
Endogenous DNA Damage
from Marnett and Plastaras, Trends Genet. 17, 214 (2001)
Biological Molecules are Labile
RNA is susceptible to hydrolysis
Reduction of ribose to deoxyribose gives DNA greater stability
N-glycosyl bond of DNA is more labile
DNA damage occurs from normal cellular operations
and random interactions with the environment
Spontaneous Changes that Alter DNA Structure
deamination
oxidation
depurination
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-46
Hydrolysis of the N-glycosyl Bond of DNA
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-47
Spontaneous depurination results in loss of 10,000 bases/cell/day
Causes formation of an AP site – not mutagenic
Deamination of Cytosine to Uracil
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-47
Cytosine is deaminated to uracil at a rate of 100-500/cell/day
Uracil is excised by uracil-DNA-glycosylase to form AP site
5-Methyl Cytosine Deamination is Highly Mutagenic
Deamination of 5-methyl
cytosine to T occurs rapidly
- base pairs with A
5-me-C is a target for
spontaneous mutations
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-52
Deamination of A and G Occur Less Frequently
A is deaminated to HX – base pairs with C
G is deaminated to X – base pairs with C
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-52