DNA Replication notes (pro- and eukaryotic)

 The most complex organisms which use DNA replication as a genomic copying mechanism
are eukaryotes, and they must condition the initiation of this important mechanism from
multiple origins in each genome and tightly control it in order to maintain genomic integrity.
This not only ensures the genetic continuity of the cell, but also of the whole organism. In
eukaryotes, this is especially challenging since the regulation of DNA regulation has to be in
sync with the inheritance of chromatin, developmental patterning tissues as well as cell
division to guarantee a single replication of the genome per cell division cycle.
 Replisomes are complex multi-enzyme machines which are responsible for the copying if
the genome.
 Problems with replicating a large amount of DNA:
1. DNA is wrapped around histones, which presents a compaction of DNA length of
about 2 million-fold.
2. Proteins associated with the DNA have to be copied as well
3. Regulation of these replication processes are complex due to the fact that each
component has to be copied once per cell cycle
 Errors in the regulation of the replication processes usually lead to accelerated rates of
mutation, which are concomitant with increased likelihood of cancer.
 Arguably, the machinery that actually carries out the copying, which includes the proteins
which initiate the entire process and the ones which use a single DNA template to produce a
duplex, are central to the whole concept of DNA replication.
 DNA repair mechanisms are superimposed on the fundamental processes of replication.
 Proteins which ensure that histones and their modifications are inherited are associated
with the DNA repair apparatus.
 Cohesin and Condensin cooperate with the replication apparatus to ensure that sister
chromatids are tethered together until the cell completes duplication of all its genetic
material.
 Only by ensuring that all of these processes are harmoniously interacting with each other
can a cell have its genetic material faithfully reproduced.

The start
 Replication starts at positions on chromosomes called ‘origins of replication’.
 Bacteria often contain one origin at which two replication forks assemble and move in
opposite directions.
 E.coli forms a single replicon at a single origin to duplicate it’s 4.6 Mb genome at a rate of 1
Kb/s for each fork, which makes the replication of its entire genome occur in 30 minutes.
 Eukaryotes have multiple linear chromosomes, each with many origins due to the much
larger genomes of eukaryotes compared to bacteria, and also because their replication forks
move at much slower speeds, at about 20-60 Kb /s , which is about 20 times more slowly
than in bacteria. It would take 50 days to replicate chromosome 1 if it only had 1 origin.
 Bacterial origins are well defined sequences to which replication initiator proteins can bind.
 Eukaryotic origins are typically not well defined at the level of DNA sequence, with the
exception of yeast S. cerevisiae. They are more defined by their chromatin organization.
Many origins correspond to regions of transcriptional activity or other features which allow
access to origin-binding proteins. Origin use is a stochastic process which is dependent on
the chromatin environment and the state of the cell cycle / of development.
 Activating origins
 The initial step in order to activate origins is to employ AAA+ family of origin-binding
proteins, which generally function as multimeric machines and are universally used by
bacterial, archaeal and eukaryotic cells.
 In bacteria, AAA+ protein DnaA forms a helical filament and then binds the origin. It then
binds ATP and unwinds an A/T rich region of the origin, resulting in ssDNA bubble onto
which replicative helicases can be loaded.
 Eukaryotic origin binding protein is called ORC (origin recognition complex). ORC is
composed of 5 AAA+ related subunits and upon recruitment to chromosomal replication
start sites, ORC binds an additional factor, AAA+ Cdc6, to form a ring hexamer, which doesn’t
unwind DNA at regions to which it binds. Although origins in eukaryotes having some
conserved sequence, except for budding yeast, ORC binds origins promiscuously. Chromatin
context plays an important, but poorly understood role in the selection of ORC binding site.

Helicase loading

 Origin-binding proteins’ main objective across all organisms is to load two helicases onto
the DNA, to form two replication forks which move in opposite directions from the
origin.
 The helicase acts as a six-subunit complex which unwinds duplex DNA by encircling one
strand of parental DNA, and through ATP hydrolysis, the helicase translocates along one
of the strands, which results in the steric exclusion of the other parental strand.
 Bacterial helicase DnaB is a homohexamer that encircles ssDNA that is created by DnaA
on the lagging-strand in a 5’-3’ direction.
 Eukaryotic helicase MCM (Mini Chromosome Maintenance) complex, is
heterohexameric, known as MCM 2-7. Each subunit is encoded in a different gene with
similar sequences and all are AAA+ protein ATPases. 4,6,7 show helicases activity in
vitro, 2,3,5 are regulatory. Zn finger motifs in 2,4,6,7 are essential for the helicase
activity. Encircles the leading strand and unzips DNA in the 3’-5’ direction.
 MCM 2-7 is also different from DnaB in that it is loaded head-to-head as a double
hexamer with dsDNA passing through the hexamer channel.
 MCM 2-7 requires Cdc45 and GINS to form CMG, which is the fully active helicase, NO
Cdc45 and GINS = NO ACTIVITY.
 a DNA bubble is topologically inaccessible to the binding of a closed-ring helicase so
many DnaB proteins require the action of a dedicated deposition factor, called DnaC, to
catalyze this loading event. DnaC is also a AAA+ ATPase which uses ATP to bind DnaB in
an inactive form and along with DnaA, it loads DnaB onto the ssDNA bubble formed by
DnaA. Hydrolysis of ATP ejects DnaC, which allows the helicase to start unwinding the
duplex.
 DnaC binds to DnaB using its N-terminal extension and remodels inra- and inter-DnaB
contacts to drive ring opening and DNA binding. DnaC’s N-terminal domain interacts
with the helicase’s C-terminus to trap it in an open ring.
 In eukaryotic cells, Cdt1 brings MCM 2-7 helicase to the ORC-Cdc6 complex bound at the
origin. MCM loading triggers ATP hydrolysis by Cdc6, leading to its ejection and the
subsequent release of Cdt1. This forms a pre-replicative complex (Pre-RC), whereby
MCM2-7 surrounds duplex DNA, but remains inactive until the onset of the S phase.
 MCMs translocate DNA to the hexamer interface to force strands apart.
  Once the lagging strand on is on the outside, the helicase’s AAA+ ATPase activity
unwinds DNA by steric exclusion.

Helicase loading and activation as replication control points

 There are fundamental differences in the regulation of helicase loading and activation
between bacteria and eukaryotes.
 DnaA binding in E.coli is regulated by SeqA, which sequesters the origin and prevents
binding of DnaA
 Sequestration is dependent on the methylation state of the DNA at the origin, as SeqA can
only bind newly replicated, hemimethylated DNA.
 In E.coli that are growing optimally, reinitiation of DNA replication occurs at the origin
before the current round of replication is completed, which results in multiple chromosomes
in one cell, which eventually separate into individual cells.
 This cannot occur in eukaryotes due to the multiple origins requirement for complete
replication to happen.
 Thus, origin initiation is tightly controlled in eukaryotes.
 This is achieved by reserving initiation events to different phases of the cell cycle and
imposing regulatory processes on the mechanism of DNA replication initiation.
 Progression to a succeeding cell cycle phase occurs through the action of specialised kinases
like CDKs, synthesis of new proteins and the regulated degradation of others.
 An additional layer of replication control is present in eukaryotes due to the
compartmentalisation of the replication process to the nucleus of the cell, which allows the
exclusion of key proteins when their activity is no longer required.
 Pre-RC formation is confined to the G1 phase. The activation of the helicase is conducted by
kinases that drive the cell into the S phase, most importantly Cdc45 and GINS.
 During S phase, Cdc6 and Cdt1 are either degraded or excluded from the nucleus to prevent
further MCM2-7 loading and reinitiation in the S phase. This is known as origin licensing; the
licensing of origins by MCM2-7 loading occurs during mitotic exit and activation occurs
during S phase by Cdc45 and GINS.

Priming

 In E.coli, primase is a single subunit enzyme called DnaG.

 It transiently binds DnaB to synthesize a ~12 nucleotide primer. DnaG binding also causes
the release of DnaC.
 In eukaryotes, this is achieved by a 4-subunit complex pol alpha/primase., only after the
transition from G1 to S phase.
 MCM10 acts as a pol alpha chaperone.
 Why is DNA synthesis not initiated de novo? Because a process as important as replication
being random has the implication that the code would be highly erroneous.
 The primase is tightly associated with DNA Pol alpha, as they co-purify in chromatography
columns even under very harsh purifying conditions.
 2 subunits for pol alpha: p180 and p70 (p68 in yeast). Catalytic and regulatory, respectively.
  Primase is also made out of 2 subunits, p58(PriL) regulatory and p49(PriS) catalytic.
 Primase can count but can’t spell.
 RNA dinucleotide formed in a slow rate-limiting step. 2 RNA nucleotides bind DNA within a
cleft between the PriS and PriL subunits.
 Error rate very high, 0.01 per nucleotide polymerised.
 The pol-beta-like hinge domain in PriL underlies its counting mechanism. There are 2
different models for its action, both involving an open and a closed conformation, whichever
comes first is not known. So the length of the primer is dependent on either how far PriS can
synthesise before it closes in on PriL and forms a closed conformation or how far it can
stretch out before it reaches an open conformation.
 The low processivity of 10nt per binding event comes down to the limitation imposed by the
hinge.
 After primer synthesis, p58 transfers the primer to pol α via direct molecular handoff.
 P58 has contacts with both p49 and pol α.
 The transfer of the primer to pol α means that
a. The current primer is stopped from being elongated any further
b. The primase enters a refractory period
c. Frees up primase for more primer synthesis
 The terminus of the primer acts as a relay race baton, picked up by p180 in pol α for
elongation.
 Pol α adds on a further ~20 DNA nucletiodes of iDNA.
 Pol α lacks proofreading and has a low processivity.
 It recognizes RNA/DNA A-form hybrid and that’s how it initiates B-form DNA synthesis,
which is also a signal for termination of primer synthesis.

The replication machinery

 The process of synthesising two daughter duplex strands simultaneously from two parent
template strands is carried out by a big fuck off multiprotein replication machine or the
replisome.
 The same core components are shared by all cells
 These are:
a. DNA polymerases
b. Circular sliding clamps
c. A pentameric clamp loader
d. Helicase
e. Primase
f. Single strand binding proteins or SSBs
 The bacterial replisome is coordinated by the clamp loader, which contains three identical
tau subunits that bind three DNA polymerases III.
 The tau subunit also binds the homohexameric helicase.
 As DnaG synthesises primers, the clamp loader keeps assembling beta clamps at
primer/template junctions for use by the lagging strand Pol III’s.
 SSB facilitates this primase/polymerase switch, by binding ssDNA and allows the clamp
loader to dislodge primase from a finished primer. Thus, SSB protects ssDNA from nucleases
and facilitates elongation by Pol III.
 There is a high processivity barrier on the lagging strand, and it is overcome by the clamp
loader prying away the polymerase after synthesis of each Okazaki fragment is completed,
replacing it at a newly primed clamp-loaded site. This way not only is the processivity barrier
overcome, but also, by having two polymerases working on the lagging strand, the speed of
1 kb/s can be maintained on both strands.
 In eukaryotes, the MCM2-7 helicase encircles the leading strand instead and needs to be
associated with Cdc45 and the tetrameric GINS complex to be activated, as they stabilize the
open conformation.
 There are also different polymerases used for each respective strand, Pol epsilon for the
leading strand and Pol delta for the lagging strand, along with Pol alpha/primase for
synthesising the primer and iDNA.
 Eukaryotes have the homotetrameric Replication protein A (RPA) as the SSB, which is
functionally analogous to the SSB tetramer in bacteria.
 Similar to bacteria, RPA modulates the primase/polymerase switch, but by a less defined
mechanism.
 The polymerases are processive by the action of the ring-shaped beta clamp-like
proliferating cell nuclear antigen (PCNA) sliding clamp, which is assembled on DNA by
replication factor C (RFC) clamp loader.
 Unlike in bacteria, RFC doesn’t appear to contact the polymerases nor the helicase.
 Oligomeric structure of the clamps allows different proteins to bind the clamp
simultaneously, which is known as the ‘’toolbelt’’ hypothesis.
 This has important implications for replisome structure and function when we refer to the
fact that all cells contain low-fidelity DNA polymerases which can bypass DNA lesions called
TLS Pols (translesion synthesis polymerases)
 In bacteria there are three types of TLS Pols, and at high concentrations induced by DNA
damage, TLS Pols can bind the beta clamp and swap places with Pol III to form a TLS
replisome
 TLS Pols move much more slowly, to allow the DNA repair mechanism to fix a lesion before
the fork encounters the damage. If a lesion is encountered, it can be bypassed.