DNA Replicates by a Semiconservative Mechanism Grow cells in 15 N and transfer to 14 N Analyze DNA by equilibrium density gradient centrifugation Presence of H-L DNA is indicative of semiconservative DNA replication from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 4-29
DNA Replicates by a Semiconservative Mechanism. Grow cells in 15 N and transfer to 14 N. Analyze DNA by equilibrium density gradient centrifugation. Presence of H-L DNA is indicative of semiconservative DNA replication. from Lodish et al ., Molecular Cell Biology, 6 th ed. Fig 4-29. - PowerPoint PPT Presentation
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DNA Replicates by a Semiconservative Mechanism
Grow cells in 15N and transfer to 14N
Analyze DNA by equilibrium density gradient centrifugation
Presence of H-L DNA is indicative of semiconservative DNA replication
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-29
The 11th Commandment
The Replicon Model
from Aladjem, Nature Rev.Genet. 5, 588 (2007)
Sequence elements determine where initiation initiates by interacting with trans-acting regulatory factors
Leading strand is synthesized continuously and lagging strand is synthesized as Okazaki fragments
Mechanics of DNA Replication in E. coli
The 5’ to 3’ exonuclease activity of Pol I removes the RNA primer and fills in the gap
DNA ligase joins adjacent completed fragments
from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-9
Initiation of DNA Replication in E. coli
DnaA binds to high affinity sites in oriB
DnaC loads DnaB helicase to single stranded regions
DnaB helicase unwinds the DNA away from the origin
DnaA facilitates the melting of DNA-unwinding element
from Mott and Berger, Nature Rev.Microbiol. 5, 343 (2007)
DnaB is an ATP-dependent Helicase
SSB proteins prevent the separated strands from reannealing
DnaB uses ATP hydrolysis to separate the strands
DnaB unwinds DNA in the 5’-3’ direction
from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-8
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-12
RNA Primer Synthesis Does Not Require a 3’-OH
Primase is recruited to ssDNA by a DnaB hexamer
Coordination of Leading and Lagging Strand Synthesis
Two molecules of Pol III are bound at each growing fork and are held together by
The size of the DNA loop increases as lagging strand is synthesized
Lagging strand polymerase is displaced when Okazaki fragment is completed and rebinds to synthesize the next Okazaki fragment
from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-11
from Pomerantz and O’Donnell, Nature 456, 762 (2008)
Interruption of Leading Strand Synthesis by RNA Polymerase
Most transcription units in bacteria are encoded by the leading strand
Natural selection for co-directional collisions in the cell
from Pomerantz and O’Donnell, Nature 456, 762 (2008)
Replisome Bypass of a Co-directional RNA Polymerase
from Pomerantz and O’Donnell, Nature 456, 762 (2008)
Replication fork recruits the 3’-terminus of the mRNA to continue leading-strand synthesis
The leading strand is synthesized in a discontinuous fashion
Replisome Bypass of a Co-directional RNA Polymerase
Bidirectional Replication of SV40 DNA from a Single Origin
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-32
Replication of SV40 DNA
T antigen binds to origin and melts duplex and RPA binds to ss DNA
Primase synthesizes RNA primer and Pol extends the primer
PCNA-Rfc-Pol extend the primer
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-31
Each cell undergoes 50,000 DNA damaging events per day
Humans possess 8-10 translesion synthesis polymerases which are error prone
The Human Genome Contains Multiple DNA Polymerases
from Loeb and Monnat, Nature Rev.Genet. 9, 594 (2008)
Initiation of DNA Synthesis
from Aladjem, Nature Rev.Microbiol. 5, 588 (2007)
ORC serves as a platform for the assembly of the preRC
CDKs phosphorylate MCM components to recruit additional proteins to form the preIC
Initiation proteins are inactivated after the ori has initiated
Replication Origins in Eukaryotes
from Gilbert, Science 294, 96 (2001)
DNA replication in metazoans initiate from distinct confined sites or extended initiation zones
Selection of initiation regions occurs via reatrictions by other metabolic processes that occur on chromatin
from Blow and Dutta, Nature Rev.Mol.Cell Biol. 6, 476 (2005)
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
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
Telomeres protect chromosome ends from being processed as a ds break
End-protection relies on telomere-specific DNA conformation, chromatin organization and DNA binding proteins
from Gilson and Geli, Nature Rev.Mol.Cell Biol. 8, 825 (2007)
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
from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004)
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 recombination-dependent 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
Telomerase Extends the ss 3’-Terminus
Telomerase-associated RNA base pairs to 3’-end of lagging strand template
Telomerase catalyzes reverse transcription to a specific site
3’-end of DNA dissociates and base pairs to a more 3’-region of telomerase RNA
Successive reverse transcription, dissociation, and reannealing extends the 3’-end of lagging strand template
New Okazaki fragments are synthesized using the extended template
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-49
The Action of Telomerase Solves the Replication Problem
from Alberts et al., Molecular Biology of the Cell, 4th ed. Fig 5-43
New Okazaki fragments are synthesized using the extended template