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8/18/2008 1
Plant Molecular and Cellular BiologyLecture 6: Regulation & Initiation of
DNA Replication
Gary Peter
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Learning Objectives
1. List and explain the structure and function of origins of replication in prokaryotes and eukaryotes
2. Explain the molecular mechanisms that regulate DNA replication of the E. coli genome
3. Explain the molecular mechanisms that regulate plasmid DNA replication
4. Explain the molecular mechanisms that regulate nuclear genome DNA replication in eukaryotes
5. Explain the molecular mechanisms by which telomeres are replicated
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Origins of DNA Replication
Specific DNA sequences that are the start site for DNA replicationWhat steps must occur at the origin to initiate DNA replication?
1. Recognition of the sequence2. Melting of the DNA3. Assembly of the replisome complex4. Initiation of leading strand synthesis
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PlasmidsExtrachromsomal DNA/genomes, usually circular, with survival and propagation functions, including
Replication controlPartitioning Multimer resolutionPost segregation killing Conjugative transfer
Molecular Microbiology (2000) 37(3), 485-491
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Control of Plasmid ReplicationReplication is central to the control of a number of important plasmid properties: HOST RANGE; COPY NUMBER; INCOMPATIBILITY; and MOBILITY Some plasmids are able to replicate in a limited number of bacterial species; they have a NARROW host range. Examples are ColE1, pBR322, pUC18 plasmids which are limited to E. coliand some closely related species.Other plasmids are able to replicate in a wide range of bacterial species; they have a BROAD host range.The number of copies of a plasmid can vary from 1 (the F plasmid) to over a hundred (pUC18). This number is a property of the plasmid itself and depends on the mechanism by which it regulates its own replication.
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Plasmid Copy # Control Systems
Negative control systems inhibit the initiation of DNA synthesisThree general classes each of which depends on the kind of negative control system employed
1. Directly repeated sequences (iterons) that complex with replication inhibitor proteins (Rep)
2. Antisense RNAs that hybrize to complementary regions of an essential RNA; these are called countertranscribed RNAs (ctRNAs)
3. Both a ctRNA and protein together
Molecular Microbiology (2000) 37(3), 492-500
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Different Mechanisms of Copy Control
Plasmid Type Control Mechanisms
Col E1 – E. coli specific OriV, antisense RNA
R1 – broad host range OriR, antisense RNA, repA protein
SC101- narrow host range Iterons
ColLb-P9 Antisense RNA, RepZ,
http://www.mun.ca/biochem/courses/4103/topics/plasmids.html
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Structure of Origin Regions in Iteron-Rep Controlled Plasmids
A 100-300 bp region termed the control or inc locus contains the origin Iterons are an array of 20 bp repeats that take up ~1/2 of the originRep proteins bind to these 20 bp repeats
Molecular Microbiology (2000) 37(3), 467-476
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Autoregulatory Mechanism of Copy # Control by Iterons-RepA
Molecular Microbiology (2000) 37(3), 467-476
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Control of ColE1 Copy #: Ct RNA and Rom Protein
Replication mediated by the synthesis of a preprimer RNA (RNA II) and the hybridization of RNA II with DNA template strand at the origin of replication, cleavage of RNA II by RNase H provides a 3’ OH for DNA polymerasectRNA I is an antisense RNA complementary to part of RNA II andROM enhances the binding of ctRNA I to RNA IIRNA I/RNA II hybrid alters the 2ndary structure of RNA II preprimer inhibiting stable formation of a DNA-RNA hybrid, which inhibits replication Molecular Microbiology (2000) 37(3), 492-500
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Plasmid Incompatibility
Two plasmids in the same incompatiability group cannot coexist in the same cellPlasmid copy number control is mediated through the action of trans-actingmolecules, those from different plasmid types with the same control mechanisms could control each others replication- eventually leading to loss
Incompatibilty group Plasmids
FI F, R386FII R1
FIII Col B-K99, Col B-K166
FIV R124
IR62, R64, R483 (at least 5 subgroups)
J R391N R46O R724P RP4, RK2Q RSF1010T R401W R388, S-a
http://www.mun.ca/biochem/courses/4103/topics/plasmids.html
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Regulation of DNA Replication in E. coli
ADP-DnaA to ATP-DnaA and back againHemimethylation-
hemimethylated oriC is not active for DNA initiation insuring only one round of replication
Katayama, Tsutomu (2001) Molecular Microbiology 41 (1), 9-17
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Initiation of Bacterial DNA Replication
The E. coli chromosome is circular and contains one origin of replication
OriC – 245 bp3 tandem AT-rich 13 bp repeats5 DnaA protein binding 9 bp repeats 11 GATC palindrome sequences that are methylated by DNA-adenine methyltransferase (Dam)
The initiation of leading strand synthesis is the key event
FEMS Microbiology Reviews 26 (2002) 355-374
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E. Coli DnaA: The Replication Initiator Protein
DnaA is a 52 kDa protein that is present throughout the “cell cycle”ADP- or ATP-DnaA is a sequence specific DNA binding protein that binds to 5 (13bp) recognition sites in oriCDnaA binds to membranes where acidic phospholipids can release ADP from DnaA in the presence of oriC to exchange it into the ATP bound form DnaA with ATP bound is the active form in replication
ATP-DnaA bends the DNA 40o, then more ATP-DnaAproteins are recruited through cooperative binding to low affinity sites located in the AT rich DNA which melt this region through binding, DnaA also helps load the DNA helicase (dnaB) onto each strand, which recruits DNA primase and DNA polymerase III binds to the melted DNA to start DNA replication on the leading and lagging strands
DNA A Structure
8/18/2008 PMCB Lecture 8: G. Peter 15Erzberger et al. 2002 EMBO J. 21: 4763-4773
Mutations in DNA A That Affect its Function
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Erzberger et al. 2002 EMBO J. 21: 4763-4773
Dnase I Footprinting & Gel Shift for Localizing DNA Binding Sites
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Model for DNA A Assembly at Ori C
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Alternative Models for Bacterial Initiation
FEMS Microbiology Reviews 26 (2002) 355-374
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Common Framework for Regulating DNA Replication
E. Coli plasmid YeastFEMS Microbiology Reviews 26 (2003) 533-554
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Yeast Origin of Replications: A Model Ori for Eukaryotes
Origins spaced on average 30,000bpPermits replication of a chromosome in ~ 8 min
Origin contains the binding site for the multisubunit origin of replication complex and binding sites for other proteins
Conserved Structures of DnaAand Cdc6/Orc1
8/18/2008 PMCB Lecture 8: G. Peter 22Erzberger et al. 2002 EMBO J. 21: 4763-4773
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Mechanisms of Initiation of Eukaryotic DNA Replication
A similar sequence of events is required for initiation of DNA replication in eukaryotesDue to the larger size of the genome in eukaryotes multiple origins existThe initiation proteins are normally bound to the origin across the G phases of the cell cyclePosttranslational control (phosphorylation) initiates DNA replication in the S phase coordinating the firing of multiple origins on each chromosome
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Preinitiation Complex in YeastOrigin Recognition Complex
A six subunit complex in yeast that binds to the Ori sequences
Other Pre-replicative Complex Proteins
Cdc6 binds to ORC and recruits Mcm proteins to form the pre-RCCdt1 – cdc10 dependentMCM proteins are helicases
Licensed Origin contains a fully assembled prereplication complex waiting for activitation by S-Cdk together with cdc7 kinase triggers DNA replication
Genes to Cells (2002) 7, 523–534
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Control of DNA Synthesis by S-CDKs
Initiation of DNA replication Cdc6 binds to ORC and recruits Mcm proteins to form the pre-RCS-Cdk together with cdc7 kinase triggers DNA replication by recruiting cdc45 to the MCM complex and inducing unwinding. The DNA polymerases α and δ bind to start replication
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Coordination of Activation of Replication Origins in Euchromatin First Before Heterochromatin
Pulse labeling experiments show that different regions of the chromosomes are replicated at different times
Dark regions have replicated in the window indicated at the bottom
Heterochromatin late in S phase
X chromosomes – inactivated one is replicated later than the active one
Increased histone acetylation directly causes the earlier firing of replication origins in yeast.
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Summary
Common steps in initiation of replication are:Origin of replication – short stretch of DNA sequenceRecognition of the ori by DNA binding proteinsRecruitment of other enzymes involved in DNA synthesisHelicase activity is the most important first step