Plant Molecular and Cellular Biology€¦ · Plant Molecular and Cellular Biology Lecture 6: Regulation & Initiation of DNA Replication ... an essential RNA; these are called countertranscribed

8/18/2008 1

Plant Molecular and Cellular BiologyLecture 6: Regulation & Initiation of

DNA Replication

Gary Peter

8/18/2008 PMCB Lecture 8: G. Peter 2

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


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


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


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.


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



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


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



Autoregulatory Mechanism of Copy # Control by Iterons-RepA



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


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


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


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


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


Erzberger et al. 2002 EMBO J. 21: 4763-4773

Dnase I Footprinting & Gel Shift for Localizing DNA Binding Sites


Model for DNA A Assembly at Ori C



Alternative Models for Bacterial Initiation

FEMS Microbiology Reviews 26 (2002) 355-374


Common Framework for Regulating DNA Replication

E. Coli plasmid YeastFEMS Microbiology Reviews 26 (2003) 533-554


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


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


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


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


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.


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

Plant Molecular and Cellular Biology€¦ · Plant Molecular and Cellular Biology Lecture 6: Regulation & Initiation of DNA Replication ... an essential RNA; these are called countertranscribed

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