Chromosomes are densely packed in mitosis
Fertilised Egg Product The accuracy of DNA replication is seen in the quality of the product
• Budding yeast replication origins map within such ARS elements on both chromosomal and plasmid DNA. • ARS elements comprise a short 11 bp A element or ‘ARS consensus sequence’: 5’-(A/T)TTTA(T/C)(A/G)TTT(A/T)-3’, plus flanking regions of 100 - 200 bp (‘B’ elements) that enhance origin function. ACS B1 B3 B2 Characteristics of ARSs
Which proteins bind to and define eukaryotic replication origins?
Replication origins in metazoans (somatic cells) • The structure of replication origins in higher eukaryotes is unclear. • Small extrachromosomal DNA sequences replicate poorly, even when carrying >10 kb genomic DNA known to act as origins when in the chromosome. • Replication initiates at specific regions at a characteristic time in S phase. Both place and timing may change with cell type. • Replication forks can potentially initiate at a number of different sites throughout an “initiation zone” that may extend over >10 kb.
The ‘Origin Number’ Paradox E. coli: Genome, 4 Mb = 4 x 106bp Fork rate approx. 800 bp / sec Replication time approx. 40 minutes = 2,400 secs Amount replicated by 2 forks in 40 mins = 2 x 2400 x 800 = 3,840,000 bp (~4 Mb) Eukaryotes Genome 20 Mb (yeast) up to 6,000 Mb (human) Fork rate 10 bp / sec (frog) - 50 bp / sec (mammal) Amount replicated by 2 forks in 8 hr (human cells) = 2 x 50 x 28,800 = 2,880,000 (~ 3 Mb, a 2,000-fold deficit) 46 chromosomes (human cells) - with one origin per chromosome, at least 92 replication forks gives approx. 140 Mb replicated in 8 hours (still a 40-fold deficit)
The solution: - eukaryotes replicate their chromosomes from multiple replication origins Electron micrograph showing an approx. 300 kb stretch of replicating chromosomal DNA from the yeast S. cerevisiae. Replication forks are indicated by an arrow. (Petes, Newlon, Byers, & Fangman 1974; Cold Spring Harb Symp Quant Biol. 38:9-16 ).
heavy labelling light labelling Interpretation: Before pulse I: End of pulse I: End of pulse II: The study of replication origins using DNA fibre autoradiography Protocol: a) Pulse label proliferating cells with 3H-thymidine for 5 min (pulse I) b) Dilute label to 1/5 activity for further 5 min (pulse II) c) Isolate DNA and spread on a photographic plate d) expose for 6 months e) develop and examine grains under microscope
Duration (hours) ~ 8 ~2 ~1 G1 S G2 M 2 hr 5 hr 9 hr = chase = BrdU pulse Chromosome regions replicate at different times • Protocol: a) Pulse cells, at different times, with BrdU for 1 hr. • b) “Chase”, collect chromosomes. • c) Stain with anti-BrdU antibodies. BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU late S 2 hr chase mid S 5 hr chase early S 9 hr chase
Typical somatic cell template DNA early-firing origins late-firing origins duplicated DNA Organization of replication during S phase
Early Drosophila embryo near-synchronous initiation The global pattern of origin usage can also change: eg early embryonic versus somatic cells: Drosophila somatic cell(transcriptionally active) S phase = 10 hours (600 mins); mean origin spacing = >40kb Early Drosophila embryo(transcriptionally quiescent) S phase = 3.4 mins; mean origin spacing = 7.9kb
stalled fork replication completed by other fork of pair double stall: no way of replicating intervening DNA Why so many origins? To allow sections of the genome to replicate faster? To allow different sections of the genome to replicate at different times? To prevent problems if origins do not initiate with 100% probability? Excess origins are used to lower the probability of a lethal ‘double stall’?
Facts I • Rate of progression of replication forks is fairly constant for a given organism • Forks generally stop only when they encounter an oppositely moving fork • Chromosome replication is regulated mainly through control of the initiation of new replication forks For example:- -by regulating the number and spacing of origins that fire eg. during development -by regulating the time during S phase at which different origins are activated
Facts II • In somatic mammalian cells, most inter-origin distances (replicon sizes) are between 30 - 300 kb (ie would take 5 - 50 min to replicate completely). • Some adjacent origins (“origin clusters”, typically 2 - 5 origins) initiate synchronously • Different origins / origin clusters initiate at different times during S phase Typical mammalian cell replicates 6,000 Mb in 8 hr = 6 x 109 ÷ 28,800 bp/sec ie. ~200,000 bp/sec For fork rate of 50 bp / sec = 200,000 ÷ 50 ~ 4,000 forks active at any given time in S phase
Restoration of chromatin after replication The principle chromatin assembly reactions during DNA replication. Reaction (a): parental nucleosomes are partially disrupted during DNA replication and the histones are directly transferred to the replicated DNA, reassembling into nucleosomes. Reaction (b): the assembly of new nucleosomes from newly synthesized and soluble histones is mediated by a chromatin assembly factor
Initiation of SV40 replication SV40 T antigen binds and distorts the viral origin. RP-A (‘replication protein A’) binds to the single-stranded DNA. DNA polymerase a -primase puts down an RNA primer and extends it with DNA. RF-C displaces pol a-primase and loads PCNA to establish the leading strand.
Mcm2-7 Mcm2-7 (mini-chromosome maintenance) proteins were originally identified in yeast because as mutants affecting replication origin usage. Fractionation showed them to be a key component of Licensing Factor. Highly conserved throughout eukaryotes; archaea also possess an Mcm2-7 homologue They are loaded onto DNA in anaphase and are removed from chromatin during S phase. They form a hexameric ring, capable of encircling double-stranded DNA.
Recap • Whether a cell has only one chromosome (as in prokaryotes) or has many chromosomes (as in eukaryotes), the entire genome must be replicated precisely once of every cell division. • Two general principles: (1) Initiation of DNA replication commits the cell to a further division. Replication is controlled at the stage of initiation. Once replication has started, it continues until the entire genome has been duplicated. (2) If replication proceeds, the consequent division cannot be permitted to occur until the replication event has been completed. • Replicon • the unit of DNA in which an individual act of replication occurs. • Origin • site at which replication is initiated. • Terminus • site at which replication stops.
A genome in a prokaryotic cell constitutes a single replicon; thus the units of replication and segregation coincide. • A plasmid is an autonomous circular DNA genome that constitutes a separate replicon; may show single copy control or under multicopy control. Any DNA molecule that contains an origin can be replicated autonomously in the cell. • Each eukaryotic chromosome contains a large number of replicons; each must be activated no more than once in each cell cycle. • The DNA of mitochondria and chloroplasts may be regulated more like plasmids that exist in multiple copies per bacterium.
Replicons Can Be Linear or Circular Key Concepts • A replicated region appears as an eye within nonreplicated DNA. • A replication fork is initiated at the origin and then moves sequentially along DNA. • Replication is unidirectional when a single replication fork is created at an origin. • Replication is bidirectional when an origin creates two replication forks that move in opposite directions.
Figure 15.1. Replicated DNA is seen as a replication eye flanked by nonreplicated DNA.
Figure 15.2. Replicons may be unidirectional or bidirectional, depending on whether one or two replication forks are formed at the origin.
Figure 15.3. A replicatin eye forms a θstructure in circular DNA.
Origins Can Be Mapped by Autoradiography and Electrophoresis Key Concepts • Replication fork movement can be detected by autoradiography using radioactive pulses. • Replication forks create Y-shaped structures that change the electrophoretic migration of DNA fragments.
Figure 15.5. Different densities of radioactive labeling can be used to distinguish unidirectional and bidirectional replication.
Figure 15.6. The position of the origin and the number of replicating forks determine the shape of a replicating restriction fragment, which can be followed by its electrophoretic path (solid line). The dashed line shows the path for a linear DNA.
15.4 Does Methylation at the Origin Regulate Initiation? Key Concepts • oriC contains eleven GATC/CTAG repeats that are methylated on adenine on both strands. • Replication generates hemimethylated DNA, which cannot initiate replication. • There is a 13-minute delay before the GATC/CTAG repeats are remethylated.
What feature of a bacterial (or plasmid) origin ensures that it is used to initiate replication only once per cycle? • Some sequences that are used for this purpose are included in the origin. oriC contains eleven copies of the sequence GATC/CTAG, which is a target for methylation at the N6 position of adenine by the Dam methylase (Figure 15.7). • If the plasmid is methylated it undergoes a single round of replication, and then the hemimethylated products accumulate (Figure 15.8). Hemimethylated origins cannot initiate again until the Dam methylase has converted them into fully methylated origins.
Figure 15.7. Replication of methylated DNA gives hemimethylated DNA, which maintains its state at GATC sites until the Dam methylase restores the fully methylated condition.
Figure 15.8. Only fully methylated origins can initiate replication; hemimethylated daughter origins cannot be used again until they have been restored to the fully methylated state.
Origins May Be Sequestered after Replication Key Concepts • SeqA binds to hemimethylated DNA and is required for delaying rereplication. • SeqA may interact with DnaA. • As the origins are hemimethylated they bind to the cell membrane and may be unavailable to methylases. • The nature of the connection between the origin and the membrane is still unclear.
Figure 15.9. A membrane-bound inhibitor binds to hemimethylated DNA at the origin and may function by preventing the binding of DnaA. It is released when the DNA is remethylated.
Each Eukaryotic Chromosome Contains Many Replicons Key Concepts • Eukaryotic replicons are 40 to 100 kb in length. • A chromosome is divided into many replicons. • Individual replicons are activated at characteristic times during S phase. • Regional activation patterns suggest that replicons near one another are activated at the same time.
S phase usually lasts a few hours in a higher eukaryotic cell. • Individual replicons in eukaryotic genomes are relatively small, typically ~40 kb in yeast or fly and ~ 100 kb in animal cells. The rate of replication is ~ 2000 bp/min, which is much slower than the 50,000 bp/min of bacterial replication fork movement. • A mammalian genome could be replicated in ~1 hour if all replicons functioned simultaneously. S phase actually lasts for >6 hours in a typical somatic cell, implying that no more than 15% of the replicons are likely to be active at any given moment. • Visualization of replicating forks by labeling with DNA precursors identifies 100 to 300 “foci” instead of uniform staining; each focus shown in Figure 15.11 probably contains >300 replication forks.
Figure 15.11. Replication forks are organized into foci in the nucleus. Cells were labeled with BrdU. The leftmost panel was stained with propidium iodide to identify bulk DNA. The right panel was stained using an antibody to BrdU to identify replicating DNA.
15.7 ReplicationOrigins Can Be Isolated in Yeast Key Concepts • Origins in S. cerevisiaeare short A-T-rich sequences that have an essential 11-bp sequence. • The ORC is a complex of six proteins that binds to an ARS.
Any segment of DNA that has an origin should be able to replicate, so although plasmids are rare in eukaryotes, it may be possible to construct them by suitable manipulation in vivo. This has been accomplished in yeast, although not in higher eukaryotes. • The discovery of yeast origins resulted from the observation that some yeast DNA fragments (when circularized) are able to transform defective cells very efficiently. These fragments can survive in the cell in the unintegrated (autonomous) state, that is, as self-replicating plasmids. • This segment is called as ARS (for autonomously replicating sequence). ARS elements are derived from origins of replication. • An ARS element consists of an A-T-rich region. • Figure 15.12: shows a systematic mutational analysis along the length of an origin.
Origin function is abolished completely by mutations in a 14-bp “core” region, called the A domain, which contains an 11-bp consensus sequence consisting of A-T base pairs. • This consensus sequence (called ACS for ARS Consensus Sequence) is the only homology between known ARS elements. • Mutations in three adjacent elements, numbered B1 to B3, reduce origin function. An origin can function effectively with any two of the B elements, so long as a functional A element is present. • The ORC (origin recognition complex) is a complex of six proteins with a mass of ~400 kD. ORC binds to the A and B1 elements. • There are about 400 origins in the yeast genome, meaning that the average length of a replicon is ~ 35,000 bp.
Figure 15.12. An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1-B3).