Biochemistry 201
Biological Regulatory Mechanisms: Lecture 3
January 14, 2013
INITIATING & CONTROLLING DNA REPLICATION
DISCUSSION
*** Fu, Y.V., Yardimci, H., Long, D.T., Ho, T.V., guainazzi, A., Bermudez, V.P.,Hurwitz, J., van Oijen,
A., Scharer, O.D., Walter, J.C. (2011) Selective bypass of a lagging strand roadblock by the
eukaryotic replicative DNA helicase. Cell. 146: 931-41.
REVIEWS
1) Gilbert, D.M. (2004). In search of the holy replicator. Nature Rev. Mol. Cell. Biol. 5:848-55
Review of literature on eukaryotic replication origins addressing puzzle of why they are so hard to
define in metazoans and suggesting that replicator paradigm that worked for E. coli, S. cerevisiae,
and viruses needs to be relaxed. Maybe metazoan origins are determined epigenetically by some
level of chromatin structure.
2) Kaguni. (2006). DnaA: Controlling the Initiation of Bacterial DNA Replication and More. Annu.
Rev. Microbiol. 60:351-.71.
Most recent description of the mechanisms that contribute to the block to re-initiation of oriC
replication. Not the clearest review because opposing papers are presented with little attempt to
resolve differences, but does show that there are still a lot of questions of exactly how oriC
replication control works, and a field ripe for a Bioreg proposal.
3)
Duderstadt, K.E., and Berger, J.M. (2008). AAA+ ATpases in the Initiation of DNA Replication.
Crit. Rev. Bioch. Molec. Biol. 43:163-187.
Many of the elongation and initiation proteins involved in replication are part of a large family of
ATP binding and hydrolyzing proteins called AAA+ proteins (world’s weakest acronym “ATPases
associated with a variety of cellular activities”). This review provides a nice overview of the
structure and function of AAA+ proteins involved in replication initiation.
IDENTIFYING INITIATION SITES (PHYSICALLY MAPPING ORIGINS)
6) Huberman, J.A. and Riggs, A.D. (1968). On the Mechanism of DNA Replication in Mammalian
Chromosomes. J. Mol. Biol. 32, 327.
A nucleotide incorporation assay visualized by fiber autoradioagraphy that demonstrated that
multiple initiation sites fire in concert, each leading to bidirectional replication. Elongation rates
and interorigin distances were also measured. Formally this was not a mapping of initiation sites,
because there were no physical reference points.
7) Danna KJ; Nathans D. (1972). Bidirectional replication of Simian Virus 40 DNA. Proc. Natl. Acad.
Sci. 69, 3097-100.
First physical mapping of replication origins in a nucleotide incorporation assay coupled with the
first use of restriction enzymes. We take these methods for granted now, but this was Nobel-prize
winning work. This method is limited to small genomes where one can account for all the
fragments generated by digestion of a genome.
8) Brewer, B.J. and Fangman, W.L. (1987). The localization of replication origins on ARS plasmids
in S. cerevisiae. Cell. 51, 463-71.
Development of the neutral-neutral gel system for identifying DNA fragments containing origins.
Based on idea that these fragments will contain replication bubbles while they are replicating and
that this unique structure confers a distinct migration position on this gel system. In subsequent
papers they later modified this technique so that they can determine the direction of fork movement
through fragments that do not contain origins (see Brewer, B.J. and Fangman, W.L. Science (1993)
262, 1728-31)
9) Raghuraman, M.K., Winzeler, E.A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart,
D.J., Davis, R.W., Brewer, B.J., and Fangman, W.L. (2001). Replication dynamics of the yeast
genome. Science. 294:115-121.
Replication entering the brave new world of microarrays. Average replication timing for every
chromosomal segment is monitored by DNA copy number and plotted against chromosomal
position to obtain a replication profile. Peaks indicates origins, valleys termination sites, peak
height indicates either origin timing and/or firing efficiency, peak slopes indicate elongation rates.
IDENTIFYING REPLICATOR ELEMENTS (GENETICALLY MAPPING ORIGINS)
10) Oka, A., Sugimoto, K., Takanami, M., and Hirota, Y. (1980). Replication origin of the Escherichia
coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the
information for autonomous replication. Mol. Gen. Genetics. 178, 9-20.
Cloning of oriC by shotgun method into colE1 plasmid and selecting ability of the plasmid to be
maintained because it contains a replicator.
11) Marahrens, Y. and Stillman, B. (1992). A Yeast Chromosomal Origin of DNA Replication Defined
by Multiple Functional Elements. Science. 255, 817-823.
Use of linker scanning analysis to dissect the structure of the B domain of an ARS. Previous
analyses using simple deletion analysis had resulted in very ambiguous conclusions about this
domain and the lack of sequence conservation had discouraged attempts to use point mutagenesis.
12) Aladjem, M.I., Rodewald, L.W., Kolman, J.L., and Wahl, G.M. (1998). Genetic dissection of a
mammalian replicator in the human beta-globin locus. Science. 281, 1005-1009.
An example of mapping initiation sites by PCR analysis of newly synthesized daughter strands.
More importantly, this paper combines genetic and physical origin mapping techniques to argue that
specific origin sequences actually exist in mammalian cells (a subject of debate and contention).
The genetic strategy for identifying origins has classically used autonomous plasmid replication as
the assay for origin function, but this strategy has failed dismally in mammalian systems. The
authors overcome this problem by integrating their sequences at specific loci in the mammalian
genome using site specific recombination and using PCR analysis to show that these sequences
induce new initiation sites at the integration loci. Unfortunately, further attempts to refine the
sequence analysis has proven difficult consistent with other experiments suggesting that initiation
may at best be confined to broad zones of tens of kilobases.
34) Dershowitz, A. and Newlon, C.S. (1993). The Effect on Chromosome Stability of Deleting
Replication Origins. Mol. Cell. Biol. 13, 391.
Latest in a series of papers that examine the effects of deleting replication origins from yeast
chromosome III. Deleting single origin has no demonstrable effect showing a considerable
redundancy of origins. You have to delete many origins before you see high rate of chromosome
loss. Thus, yeast origins are highly redundant.
INITIATION OF E. COLI DNA REPLICATION
13) Fuller, R.S., Kaguni, J.M. and Kornberg, A. (1981). Enzymatic replication of the origin of the E.
coli chromosome. Proc. Natl. Acad. Sci. USA 78, 7370-7374.
Breakthrough in establishing conditions in vitro for replication initiation at the E. coli origin. No
substitute for blood, sweat, and tears (lots of tears) in setting up great in vitro systems.
14) van der Ende, A, Baker, T.A., Ogawa, T., and Kornberg, A. (1985). Initiation enzymatic replication
at the origin of the Escherichia coli chromosome: Primase as the sole priming enzyme. Proc. Natl.
Acad. Sci. USA 82, 3954.
Initial dissection of the initiation reaction, starting with the description of a presynthesis step, a step
during which no nucleotide incorporation is occurring but something is happening that allows later
incorporation to occur. Uses kinetics, dNTP depletion, and temperature shifts to perturb the
reactions and identify intermediates that form during initiation.
15) Baker, T.A., Sekimizu, K., Funnell, B.E., and Kornberg, A. (1987). Extensive Unwinding of the
Plasmid Template during Staged Enzymatic Initiation of DNA Replication from the Origin of the
Escherichia coli Chromosome. Cell. 45, 53-64.
Excellent use of serendipity (or the "prepared mind") in the development of an unwinding assay to
define more precisely what DnaA, DnaB, DnaC and SSB does to the DNA during presynthesis.
These proteins cause the DNA to be highly underwound and to migrate faster than standard
supercoiled DNA on a gel.
16) Funnell, B.E., Baker, T., and Kornberg, A. (1987). In vitro Assembly of a Prepriming Complex at
the Origin of the Escherichia coli Chromosome. J. Biol. Chem. 262, 10327-10334.
Direct examination of DnaA and DnaB (and not DnaC) protein on the origin by immunoEM.
17) Baker, T.A., Funnell, B.E., and Kornberg, A. (1987). Helicase Action of DnaB Protein During
Replication from the E. coli Chromosomal Origin in vitro. J. Biol. Chem. 262, 6877.
Complex loading and bidirectional unwinding (with DnaB at the ends of the bubbles) seen directly
by EM.
*18)
Bramhill, D. and Kornberg, A. (1988). Duplex Opening by DnaA Protein at
Novel Sequences in
Initiation of Replication at the Origin of the E. coli Chromosome. Cell 52, 743.
Shows that DnaA protein bound to ATP is sufficient to open the DNA duplex at oriC. SS vs DS
specific probes are used to monitor the unwinding at oriC and to demonstrate that the duplex is
opened at the 13 bp AT-rich repeats.
19) Speck, C. and Messer, W. (2001). Mechanism of origin unwinding: sequential binding of DnaA to
double- and single-stranded DNA. EMBO J. 20, 1469-76.
Systematic analysis of DnaA-ATP binding to double and single-stranded motifs in oriC leads to a
proposal for how DnaA-ATP can unwind oriC using just binding energy.
20) Fang, L., Davey, M.J., and O’Donnell, M. (1999). Replisome Assembly at oriC, the Replication
Origin of E coli, Reveals an Explanation for Initiation Sites outside an Origin. Mol. Cell 4, 541533.
Targeted mutagenesis of ATP hydrolytic activity of DnaB helicase allows O’Donnell’s group to
examine the loading of DnaB without having to worry about its helicase activity. Protection assays
on the unwound single-stranded DNA shows that two DnaB helicase complexes are initially
positioned in a manner that requires them to move past each other to get to their respective forks.
*21)
Davey, M.J., Fang, L., McInerney, P., Georgescu, R.E., and O’Donnell, M.
(2002). The DnaC helicase loader is a dual ATP/ADP switch protein. EMBO J. 21, 3148-59.
Careful analysis of role of ATP binding and hydrolysis by DnaC in the binding, loading, and
inhibition of DnaB. This is a beautiful example of how the power of biochemistry in dissecting a
mechanisms can be greatly enhanced by the use of targeted genetics. Key to this paper was having
special mutants in the ATP binding pockets of DnaB and DnaC, allowing the role of these pockets
to be distinguished. The hydrolysis of DnaC acts as a switch, but it is not simply switching between
an active and inactive form. The ATP bound form is active for early functions in the loading cycle,
but inhibitory for later functions. The ADP bound form is inactive for the early functions, but
permissive for the later functions. The hydrolysis ensures that both forms act in the proper order to
execute the loading of DnaB.
CONTROL OF ORIC INITIATION DURING THE E. COLI CELL CYCLE
22) Helmstetter, C.E. and A.C. Leonard (1987). Coordinate Initiation of Chromosome and
Minichromosome Replication in E. coli. J. Bacteriol. 169, 3489.
Demonstration that small plasmids that replicate using oriC fire simultaneously with the
chromosome origin. Shows that "initiation potential" is restricted to brief windows of time in the
bacterial cell cycle.
23) Russell, D.W., and N.D. Zinder (1987). Hemimethylation prevents DNA replication in E. coli. Cell
50, 1071-1079.
Indirect evidence that hemimethylated oriC DNA is refractory to initiation. Key was synthesizing
and presenting templates of defined methylation state to cells via transformation. Transformation
frequency is only an indirect assay for replication, so the title is a bit overstated, but the hypothesis
they generated was a good working model that proved true.
24) Boye, E. and Lobner-Olesen, A. (1990). The Role of dam methlytransferase in the control of DNA
replication in E. coli. Cell 62, 981.
Demonstration that if the level of dam methylase in the cell is too high or too low, initiation of
replication at the multiple oriCs in a cell occurs asynchronously. In other words the number of
oriCs in a cell deviates from the expected numbers of 1, 2, 4, 8, or 16. Copy number of oriC is
counted by simultaneously inhibiting replication initiation and cell division, letting the replication
forks complete replication, and counting the total number of genomes by flow cytometry. Cells
with 3, 5, 6, or 7 genomes are scored in an asynchrony index. This is a very indirect assay for
uncontrolled initiation, but was the first genetic evidence that dam methylase might contribute to the
control of replication initiation.
*25) Campbell, J.L. and Kleckner, N. (1990). E. coli oriC and the DnaA gene promoter are sequestered
from the dam methyltransferase following the passage of the chromosomal replication fork. Cell 62,
967.
Experimental demonstration using synchronized cell cultures that oriC appears to be protected from
remethylation soon after replication initiation. Key is using methylation sensitive restriction
enzyme to monitor hemimethylation state of one of the oriC GATC sites. Led to the idea that
hemimethlyated oriC is sequestered from dam methylase and that a similar sequestration from
initiation proteins could help prevent immediate re-initiation. What that sequestration ex
26) Landoulsi, A., Malki, A., Kern, R., Kohiyama, M., and Hughes, P. (1990). The E. coli cell surface
specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates.
Cell. 63, 1053-1060.
E. coli outer membrane preps, which were first shown by Schaecter’s group to bind specifically to
hemimethylated oriC, are now shown to specifically inhibit in vitro replication of hemimethylated
oriC templates. Inhibition appears to be through preventing DnaA binding to oriC. A potential
assay for purifying the putative sequestration activity, but the biochemists were beat out by the
geneticists (see Lu et al. #29).
27) Lu, M., Campbell, J.L., Boye, E., and Kleckner, N. (1994). SeqA: a negative modulator of
replication initiation in E. coli. Cell. 77, 413-426.
Kleckner's group hypothesizes that the failure of hemimethylated DNA to replicate is due to active
inhibition of oriC rather than a failure to activate oriC. They devise a genetic screen for loss of
function mutations that allow methylated DNA to transform dam(-) strains efficiently, using the
Russell and Zinder presumption that the failure to transform is due to a failure to replicate. This
leads to seqA. Mutation impairs sequestration of hemimethylated oriC and disrupts initiation
synchrony, but cells are viable and the most significant effect observed is asynchrony of initiation.
Even later direct analysis of DNA content by flow cytometry (PNAS 1996. 93:12206-211), showed
only a slight propensity to over-replicate. Clearly not the entire story.
28) Nievera, C., Torgue, J.J., Grimwalde, J.E., and Leonard, A.C. (2006). SeqA blocking of DnaA-oriC
interactions ensures staged assembly of the E. coli pre-RC. Mol. Cell. 24:581-92.
Translating the general concept of "sequestration" to a specific biochemical mechanism has been
difficult and controversial. In this paper the authors monitor DnaA binding in vivo by genomic
footprinting. They argue that SeqA allows DnaA to bind its high affinity sites on the
hemimethylated oriC but prevents DnaA from binding its low affinity sites where origin unwinding
occurs.
*29)
Sekimizu, K., Bramhill, D., and Kornberg, A. (1987). ATP activates DnaA protein in initiating
replication of plasmids bearing the origin of the E. coli chromosome. Cell. 50, 259-265
Discovery that ATP has an allosteric role in activating DnaA for initiation. Moreover, the ADP
bound form is inactive for initiation and the two forms can interconvert by slow hydrolysis and slow
nucleotide exchange. Raises the possibility of a regulatory role for ATP/ADP in restricting DnaA
function in the cell to a brief window of the cell cycle (see ref 24).
30) Kurokawa, K., Nishida, S., Emoto, A., Sekimizu, K., and Katayama, T. (1999). Replication cyclecoordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherchia coli.
EMBO J. 18, 6642-52.
Nucleotide bound state of DnaA in vivo fluctuates in synchronized cell populations, consistent with
this state helping to regulate DnaA initiation potential. Extremely tight binding affinity of ATP and
ADP for DnaA allowed IP of DnaA and analysis of bound nucleotide. Regeneration of the ATP
bound "active" state of DnaA requires new protein synthesis consistent with an old observation that
each new round of initiation requires new protein synthesis. Suggests that synthesis of new DnaA
(which would bind ATP, the most abundant nucleotide in the cell) is the primary means of
regenerating active DnaA. However, in other papers Kornberg's lab explored the stimulation of
DnaA nucleotide exchange by phospholipids suggesting a second potential pathway for regenerating
active DnaA that involves membrane association. Regeneration of DnaA-ATP is still an open
question.
*31) Katayama, T., Kubota, T., Kurokawa, K. Crooke, E., and Sekimizu, K. (1998). The initiator
function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal
replicase. Cell. 94, 61-71.
Intrinsic ATP hydrolysis of DnaA in the allosteric ATP binding site is very slow, but can be greatly
stimulated by an activity detected in crude extracts. When this activity is purified it separates into 3
factors, one of which is purified and turns out to be the beta subunit (clamp). The other two
requirements are an unpurified factor called IdaB and clamp loading activity (provided by gamma
complex or PolIII* holoenzyme). For the beta subunit to stimulate DnaA's ATP hydrolysis it must
be loaded onto DNA by the clamp loader. Provides a potential means of coupling initiation of DNA
synthesis to inactivation of DnaA for re-initiation.
32) Kato, J. and Katayama, T. (2001). Hda, a novel DnaA-related protein, regulates the replication cycle
in Escherichia coli. EMBO J. 20, 4253-62.
Further purification of IdaB activity was very difficult, and it was genetics that led to its
identification. Taking a page from the eukaryotic replication field, the authors looked for new
replication mutations by looking for elevated plasmid loss. One of these mutants (in PCNA) was
suppressed by high copy Hda. Hda is detectable in the IdaB fractions and large quantities of
purified Hda can substitute for IdaB in the biochemical assay that inactives DnaA-ATP .
Conditional (ts) mutants of Hda show subtle signs of over-initiation when shifted to restrictive
temperatures. Consistent with Hda having some but not the entire responsibility of preventing reinitiation. No one has yet combined the hda and seqA mutants in a conditional manner.
33) Nishida, S., Fujimitsu, K., Sekimizu, K., Ohmura, T., Ueda, T., and Katayama, T. (2002). Negative
control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of
chromosomal DNA replication in Escherichi coli. J. Biol. Chem. 277, 14986-95.
Targeted mutagenesis of the ATP binding pocket of DnaA generates a mutant with normal ATP
binding and initiation activity but defective ATP hydrolysis in vitro and in vivo (authors are lucky
since this is a difficult mutation to obtain). Overexpression of this mutant protein induces more reinitiation than overexpression of wild-type DnaA, suggesting a role for ATP hydrolysis in
preventing re-initiation.
SOME QUESTIONS TO THINK ABOUT THE LECTURE
1) Could you use electron microsopy to determine the site of initiation? Would it work for small
genomes or large? What would you use as a physical reference point? Could you distinguish
bidirectional replication from unidirectional replication?
2) Can you map the site of replication initiation by incorporation of radioactive precursors. How would
you do it for small genomes (like a eukaryotic virus under 50 kb). How would you do it for a cellular
genome?
3) The bubble arcs shown in the handout are for fragments which have origins precisely in the middle.
What would you see if the origin were asymmetrically placed in the fragment? How would you modify
the neutral-neutral gel assay to determine the direction of fork movement in a fragment being replicated
by an outside origin. (Hint: assume you can diffuse in restriction enzymes into the gel between the first
and second direction)
4) Why does the 2-D gel assay for replication initiation require a Southern blot? What is its resolution,
i.e. how precisely can it map origins of replication?
5) Why has the genetic approach for identifying replication origins traditionally depended on assaying
replication or maintenance of an autonomous plasmid? Why might this approach have failed in
mammalian cells?
6) How might you identify initiation sites for bidirectional replication by mapping okazaki fragments?
7) How does a consensus site provide information about the functional relevance of specific nucleotides?
How might a consensus site miss the functional relevance of specific nucleotides?
8) How would you assay the effect of DnaB and SS DNA association on DnaC ATPase activity? Keep
in mind that DnaB has a very strong ATPase activity on its own that can mask the ATPase activity of
DnaC. This activity is inhibited when DnaB binds to DnaC but is released when DnaB is loaded onto SS
DNA by DnaC.
9) Constitutive dam+ and dam- bacteria both grow and replicate reasonably well. Why would the dambacteria do so well even though they cannot be readily transformed by methylated or hemimethylated
plasmids? What would you anticipate to be the phenotype of a tight dam- conditional mutant suddenly
shifted to the restrictive temperature?
10) How would you determine whether new synthesis of DnaA is required for each round of initiation?
POINTS RAISED IN LECTURE
1) Molecular biology allows functional characterization of sequence elements in vivo. The
molecular biology revolution was made possible by : (1) the capacity to isolate and clone specific DNA
sequences from a cell’s DNA or RNA; and (2) the capacity to functionally assay these sequences (and
mutant derivatives) in the cell. The first capacity arose from the ability to cut (restriction enzymes) and
paste (ligase) DNA segments into special cloning vectors and made it possible to alter segment
boundaries and sequence. The second capacity arose from the ability to reintroduce sequences back into
cells (i.e. transformation) and to assay their function in vivo (e.g. reporter genes).
2) Fine mapping sequence elements: the power and limitation of consensus sites. Mutagenesis of
a sequence element allows a direct test of the functional importance of individual nucleotides.
Consensus sites, sequences that are evolutionarily conserved in a class of genetic elements across or
within species, provide a window on the extensive mutagenesis experiments performed by nature and
suggest sequences that are functionally important for that class.
There are two important limitations to keep in mind. First, consensus sites only identify sequences
that are functional important for the entire class of genetic elements (i.e. the “lowest common
denominator” function); sequences that might be important for a subclass of elements or individual
elements can be missed in this comparison. Second, some of the functions that nature selects for may
not be detected in the assays being used. Hence, the functional importance of some consensus sites
might not be apparent with available assays.
3) Origins can be mapped genetically or physically. Origins were first conceived by Francois Jacob
as genetic elements that allowed a DNA molecule to be replicated in cis, and he called them
“replicators”. Replicators are operationally defined as sequence elements that confer autonomous
maintenance on a plasmid. Such an operational definition does not presume anything about the
mechanism of replication, but (1) requires that the sequences be sufficient for plasmid maintenance and
(2) assumes the sequence behaves the same in a plasmid as it does in the genome. To avoid such an
assumption, yeast replicators were called autonomously replicating sequences (ARSs) for a long time
before they were proven to act as origins in the genome.
Origins can also be physically mapped as sites where replication initiates. Physical mapping
techniques can be divided into two fundamental classes: (1) identifying a converging series of nested
replication intermediates based on their distinctive structure (e.g. replication bubbles); or (2) identifying
the site of new DNA synthesis based on nucleotide incorporation or increased copy number. The first
class relies on assumptions or knowledge about the mechanism of replication and the nature of the
replication intermediates. Initiation usually (but not necessarily) begins within or very near replicators,
allowing the genetic and physical definitions of origins to overlap.
Recently a combination of these two definitions has been used to try to identify elusive metazoan
origins (see below). Investigators are trying to define genetic elements that, when integrated in the
genome, are sufficient to confer new sites of replication initiation.
4) Eukaryotes contain multiple redundant origins. The large expansion in the size of eukaryotic
genomes depended on the evolution of multi-origin genomes so that large genomes could be replicated
in a reasonable amount of time. Eukaryotic cellular origins initiate bidirectional replication with each
fork colliding with converging forks from neighboring origins. Thus, interorigin distances, which
generally range from 30 kb to 200 kb, determine the region replicated by each origin (i.e. the replicon).
If an adjacent origin does not fire, however, forks continue further until it collides with a converging
fork from the next origin over. In S. cerevisiae several origins in a role can be deleted without seriously
compromising chromosome replication, illustrating the redundancy of origin usage throughout most
eukaryotic genomes.
5) Chromatin structure and function contribute to origin specification in eukaryotes. The best
defined eukaryotic origins are from S. cerevisiae or from eukaryotic viruses. All these origins have
consensus sequences that provide specific binding sites for initiator proteins that carry out origin
recognition (see initiation tasks below) and can be readily identified using ARS assays. Most also have
transcriptional elements that strongly enhance a core origin sequence (not unlike transcriptional
enhancement of basic promoter elements) or are in fact critical elements of the origin (see initiation on
chromatin below). In a few cases, these transcriptional elements have been shown to help the replication
machinery deal with the chromatin structure coating eukaryotic DNA. Not all ARSs identified on
plasmids act efficiently as origins at their endogenous locus in the chromosomes. This suggests that, in
addition to initiator sequence recognition, poorly defined features of chromatin structure also help
specify origins.
Origins from multicellular eukaryotes have been harder to define, as they don’t register in ARS
assays and can physically map to broad regions of the genome. Moreover, the lack of strong sequence
specificity displayed by the initiator protein ORC, suggests that chromatin structure and function play
the primary role in specifying origins. Consistent with this notion is the observation that initiation
occurs nonspecifically in large zones (10s of kb) of initiation. These are often regions of the genome
free of any interfering transcriptional activity or chromatin structure.
6) The initiation machinery must carry out five tasks: (1) recognition of the origin; (2) localized
unwinding at the origin; (3) loading the replicative helicase; (4) priming the continuous strand; (5)
loading the DNA synthetic machinery. Having an elaborate loading procedure for helicases ensures that
they do not accidentally unravel duplex DNA when they are not needed and that the right helicase is
used for each job. Of course, this means that if a fork runs into problems and collapses (which happens
more frequently than the field originally appreciated), an elaborate procedure is needed to reload the
helicase and restart the fork. In some sytems, other methods have evolved to begin new DNA synthesis
without de novo synthesis from a primase (e.g. protein priming for adenovirus and tRNA for
retroviruses).
7) E. coli initiates DNA replication using three AAA+ ATPases, DnaA, DnaB, and DnaC. The
DnaA protein specifically binds the origin of replication at multiple sites and, when complexed with
ATP, can partially unwind an AT rich segment of the origin (apparently by cooperatively spreading out
from the duplex binding sites and preferentially binding one single strand of the AT rich segment). The
replicative helicase (DnaB) is then loaded onto the partially melted DNA by DnaC protein. Analagous
to the loading of sliding clamps, the loading of the toroidal DnaB hexamer appears to require a ringopening, mediated by binding to six DnaC-ATP molecules, and a ring closing (over the unwound SS
oriC DNA instead of a primer-template duplex), mediated by DnaC ATP hydrolysis, which releases
DnaC from DnaB. In this manner two DnaB hexamers are loaded, one on each strand of an unwound
origin. This loading does not require the DnaB’s ATPase activity. However, once loaded, DnaB
ATPase activity (and presumably helicase activity) is needed for the helicases to move past each other
toward their respective forks so as to make room for primases to lay down primers. The resulting
primer-template structures then allow a clamped PolIII holoenzyme to initiate leading strand synthesis at
each fork.
8) Partial reactions for E. coli replication reaction were identified by several methods discussed at
the beginning of my second replication lecture: (1) kinetic analysis to separate the reaction into a slow
presynthesis and a fast synthesis step; (2) using the temperature dependence of an early step to divide the
reaction into two stages where only the early step occurs, then only the late step occurs; (3) removing
proteins or cofactors (like dNTP or rNTPs) from the reaction; (4) using ATPS to block steps dependent
on ATP hydrolysis; (5) using mutant proteins that have specific functions blocked. The nature of the
partial reaction was characterized by applying different techniques (gel mobility, EM, immunoEM,
differential sensitivity of DS and SS DNA to nuclease and chemicals) to isolate and analyze the
intermediates that are formed. Some examples are outlined in the appendix of this handout.
9) E. coli initiates exactly once per cell cycle in a specific window of the cell cycle during steady
state growth. During rapid growth, cell division intervals are shorter (20 min) than the time required to
replicate the genome (minimum of 40 min). The solution is to spread a single full round of replication
over more than one division cycle. For any particular cell cycle, a replication cycle is terminated,
generating the two genomes that will be segregated when the cell divides, but that replication cycle was
initiated in a previous cell cycle. Also in that same cell cycle, a new replication cycle is initiated, but it
won’t be completed until a later cell cycle, and occurs on a genome that is already in the midst of
replication. Thus, during rapid steady state E. coli doubling, genomes contain replication bubbles within
replication bubbles, and genomes are segregated while they are actively replicating, in contrast to
eukaryotes that confine replication and segregation to mutually exclusive cell cycle periods.
Importantly, although a new round of replication can be initiated before previous rounds are
terminated, within each cell cycle there is still only one synchronous round of initiation events (for
eventual termination and segregation in a subsequent cell cycle) and one termination event (of a round of
replication that initiated in a previous cell cycle). Thus, E. coli replication is still highly regulated so that
it is initiated once and only once in a restricted window of each cell cycle.
10) Two mechanisms for preventing re-initiation within the E. coli cell cycle have been identified
so far. One involves the preferentially binding of SeqA to hemimethylated (and hence newly replicated)
oriC. Exactly how this binding prevents origin unwinding is unknown, but it does not seem to occlude
DnaA from origin sequences as was originally suspected. This mechanism is ideally suited for an
immediate block to re-replication, as the act of replication activates the block by converting fully
methlyated parental DNA to hemimethylated daughter DNA. However, for reasons that are not
understood, the SeqA mediated block only lasts for a third of the cell cycle, so it cannot account for the
full cell-cycle block to re-initiation
The second mechanism involves the conversion of DnaA from an active ATP bound state to an
inactive ADP bound state by an intrinsic weak hydrolytic activity of the protein. This activity is greatly
stimulated by a regulatory protein, Hda, but only when it is bound to a chromosome-loaded beta clamp.
Such a dependency is thought to couple DnaA inactivation to completion of replication initiation (i.e.
loading of the clamp onto the leading strand primers). This Hda-mediated mechanism can remain to
inhibit re-initiation when the SeqA mechanism is phased out. But it raises the question of how and when
active ATP-bound DnaA is regenerated, since intrinsic release of ADP is very slow. Both synthesis of
new DnaA and phospholipid (i.e. membrane) stimulated ADP release have been suggested as possible
solutions, but the exact mechanism DnaA-ATP regeneration and how it is coupled to the cell cycle
remains a mystery (and good Bioreg proposal topic).