1
MB3005
DNA REPLICATION
AIMS:
To review:
1 supercoiling;
2 origins;
3 the ‘end problem’.
Refer to
BI20M3 Lectures on DNA Replication
Lodish, (6th edition, 2008 Chapters 4, 10).
http://www.abdn.ac.uk/~bch118/index.htm
2
1
THE NATURE OF SUPERCOILING
A Watson-Crick double-helix has about
10.5 base-pairs per turn:
In this form,
bases in base-pairs
are directly opposite one another,
linked by H-bonds;
rings of bases on each strand
are stacked on top of each other,
stabilised by hydrophobic interactions.
The structure
stable.
is
thermodynamically
3
In cells, however, most DNA is underwound.
Enzymes (pp. 20-22)
break, unwind and re-seal DNA,
so that it has >10.5 base-pairs per turn.
Why does underwinding produce
>10.5 base-pairs per turn?
Consider a length of double-helical DNA:
4
So, after underwinding,
there is the same length of DNA
(i.e. number of base-pairs), but less turns.
Thus, there is an increase in base-pairs per turn.
5
When underwinding occurs,
H-bonds of base-pairs are strained:
and bases are less stacked,
so the structure is less stable.
6
One way in which stability can be
regained is for the underwound
structure to twist (‘writhe’).
Consider an
double-helix.
underwound
It has >10.5 bp/turn
is ‘flat’
is unstable:
By twisting,
it reverts to 10.5 bp/turn
is ‘supercoiled’
regains stability.
circular
7
The result is a
plectonemic (twisted thread)
right-handed (like the turn of the doublenegative
helix)
(compensates for underwinding)
supercoiled structure.
8
2
SUPERCOILING AND DNA
PACKAGING
Isolated circular double-stranded DNA
usually has plectonemic supercoils
(Lodish, p. 107).
9
The prokaryotic genome is a ‘nucleoid’
of plectonemic DNA attached to a
protein core:
Nucleoid cross-section
(in part)
10
Underwinding also occurs in
linear double-helical DNA.
It might be expected that any
underwinding could be relieved simply
by the ends of the two strands rotating
around each other,
so that stability is regained,
and no supercoiling need occur.
In fact, eukaryotic linear DNA behaves
instead as a closed structure
(i.e. rather like circular DNA)
because, in chromatin, it is bound to
protein.
So,
when linear DNA is underwound,
supercoiling does occur.
11
Linear DNA supercoiling is more
compact than the plectonemic form;
it wraps around protein
chromatin nucleosomes.
to
form
The result is a
solenoidal
left-handed
negative
(like a telephone cord)
(compare with plectonemic form)
supercoiled structure.
12
As before, the instability caused by
underwinding is relieved.
Cells, then,
purposefully underwind DNA,
so that the resulting supercoiling
forms structures convenient for
DNA packaging.
13
3
SUPERCOILING AND INITIATION
OF DNA REPLICATION
Returning to the underwound circular
double-helix (p. 6):
It has >10.5 bp/turn
is ‘flat’
is unstable.
Stability may
be regained
by supercoiling.
14
Stability may also be regained
if the two DNA strands partially separate,
causing the remaining double-helix to return to
10.5 bp/turn.
15
The two structures:
(a) supercoiled;
(b) partially separated
are interconvertible.
So:
negative supercoiling
(of circular or linear DNA)
not only allows compact packaging:
it also provides a structure
prone to partial strand separation.
Such separation, of course,
is needed for DNA replication to begin.
So:
DNA is stored in a form
energetically activated
for local unwinding
needed at DNA origin(s) of replication.
16
4
HOW SOLENOIDAL STRUCTURES
FORM
Recollect that linear DNA forms solenoidal
supercoils (p. 11).
Oddly, eukaryotic cells lack enzymes that
underwind DNA,
so how do the solenoids form?
Answer:
although not underwound,
linear DNA wraps around nucleosome
proteins
in negative solenoidal supercoils.
These are then compensated for
when DNA that is not wrapped
twists in the opposite direction,
forming positive supercoils (p. 18)
that are later removed.
17
It is easiest to visualise this for circular
(rather than linear) DNA binding to a
protein:
18
5
POSITIVE SUPERCOILING
This occurs when DNA is overwound.
DNA twists in the opposite direction to
that
of
underwound,
negatively
supercoiled DNA.
19
Positive supercoils form in front of
replication forks
as parental strands are pushed apart,
and are removed as they form.
They also occur in some thermogenic
microbes.
Recollect that underwound DNA
is prone to partial strand separation
(pp. 14-15).
Conversely, in overwound DNA,
strand separation is more difficult.
Perhaps overwinding prevents strand
separation that would otherwise occur
at high temperature.
20
6
ENZYMES OF SUPERCOILING
The enzymes involved,
topoisomerases,
occur in all DNA-containing cells.
Type I:
break one DNA strand;
swivel broken end around
intact strand;
re-seal.
Type II: break both strands;
pass intact strand through
gap;
re-seal.
Most remove supercoils;
a few introduce supercoils.
Supercoil introduction needs ATP.
(‘DNA is … energetically activated …’ p. 15).
21
In E. coli,
DNA is kept appropriately negatively
supercoiled
by co-ordinated activities of:
DNA gyrase (a type II)
(which introduces negative supercoils)
and
a type I
(which removes negative supercoils).
22
The relevance of supercoiling in DNA
replication (pp. 14-15) is emphasised by
clinical use of topoisomerase inhibitors:
Novobiocin, nalidixic acid
(inhibit DNA gyrase)
are widely used antibiotics;
Camptothecin
(inhibits eukaryotic type I)
is an antitumour agent.
23
MB3005
DNA REPLICATION
AIMS: To review:
origins
and the initiation of DNA replication.
Refer to
BI20M3 Lectures on DNA Replication
Lodish, (6th edition, 2008 Chapters 4, 10).
http://www.abdn.ac.uk/~bch118/index.htm
24
1
RECAPITULATION
Prokaryotic circular DNA has a single
origin
and
eukaryotic linear DNAs have multiple
origins,
at which parental strand separation
occurs
and
from
which
bidirectional
replication begins.
(BI20M3)
25
2
THE E. coli ORIGIN (OriC)
This is the best studied origin.
It was identified by
inserting random restriction fragments
of E. coli DNA into a plasmid lacking an
origin.
Some engineered plasmids were able to
replicate in the test-tube using purified
E. coli DNA replication proteins.
They must contain an E. coli-derived
origin.
OriC is a 245 base-pair segment,
containing sequences
highly conserved in related bacteria.
26
These include:
4
3
copies of a
copies of a
9-mer sequence
13-mer sequence
rich in A.T.
To the left (as drawn)
is another A.T-rich region.
There are also 8-14 GATC sequences:
these are sites of action of
deoxyadenosine methylase (DAM).
(p. 39)
27
For initiation at OriC:
A DnaA binds.
~20 copies
sequences,
bind to
the
9-mer
helped by HU protein.
To bind, DnaA must have ATP
attached. (p. 39)
B DnaA opens the 13-mer sequences.
This only occurs if the DNA is
negatively supercoiled.
A.T content of 13-mers aids opening.
C DnaB binds,
helped by DnaC.
It unwinds DNA bidirectionally,
forming two replication forks
(i.e. it is a helicase).
28
D Many copies of SSB protein
bind co-operatively to separated
strands,
preventing duplex re-formation.
E DNA gyrase (p. 21)
removes positive supercoils ahead of
the replication forks. (p. 19)
F Primase initiates leading
(and later lagging) strand synthesis.
(BI20M3)
29
3
THE SV40 ORIGIN
SV40 is simian virus 40.
It contains small, double-helical circular
DNA
with a single origin.
The virus uses host (eukaryotic) cell
enzymes
and just one virus-coded protein
(‘T antigen’)
to replicate its DNA.
30
Its origin is a 65 base-pair segment with
three regions:
T antigen, as two hexamers,
binds to the middle region,
and
unwinds DNA bidirectionally,
through the A.T-rich regions,
forming 2 replication forks
(i.e. it is a helicase).
31
So,
T antigen reproduces activities of
E. coli
DnaA
and DnaB
(initiator)
(helicase).
T antigen activity may be controlled by
its phosphorylation state.
32
4
THE ORIGINS OF YEAST(S)
These have been identified in a similar
way to that used for OriC:
Random restriction fragments of yeast
DNA were circularised (i.e. made into
plasmids) and inserted into yeast cells;
some
were
able
to
replicate
autonomously (i.e. independently of the
cell DNA):
they must contain a yeast-derived
origin.
They are called
‘autonomously-replicating sequences’
(ARSs).
There are about 400,
spread through the
17 yeast chromosomes.
33
ARSs are ~150 base-pair segments with
four regions:
The following events initiate DNA
replication:
A 6 proteins form
an origin-recognition complex (ORC)
which binds B1/A.
B After a cell has divided,
protein ‘licensing factors’ (p. 38)
bind to ORC
and ‘license’ the cell to begin a new
round of DNA replication.
34
C ARS-binding factor 1
binds to B3 and causes
strand separation at B2.
(So, B3 and B2
are roughly analogous to
OriC 9- and 13-mers respectively.)
D Helicase
and other replication proteins
bind.
35
5
THE ORIGINS
EUKARYOTES
OF
HIGHER
The method used to identify yeast ARSs
(p. 32)
has been mostly unsuccessful
in the search for origins of
higher eukaryotes
Some mammalian ‘plasmids’,
inserted into yeast cells, do replicate,
but apparently only because, by chance,
they have a sequence
similar to that of a yeast ARS.
They seem not to be mammalian cell
origins.
36
At present, what we can say is:
(i)
homologues of yeast ORC occur in
all eukaryotes examined;
(ii)
DNA-bound ORC recruits other
proteins, including a helicase,
to form a ‘pre-replication complex’;
(iii)
on S phase entry,
protein kinases activate the complex,
leading to strand separation and
DNA polymerase binding.
In a few origins,
ORC binds to defined sequences,
e.g. the origins near the human lamin B2
gene and the human -globin gene cluster.
But, elsewhere, origins seem to consist of
particular, co-operating regions, that
perhaps spread over long stretches of DNA,
and seem to be selected by ORC to
different extents in different circumstances.
37
Factors affecting ORC binding to a region
of DNA may include:
(i)
transcriptional activity of the DNA;
(ii)
methylation of the DNA;
(iii) acetylation of histones around the DNA.
Also,
perhaps ORC is itself recruited by other
proteins,
that have previously bound to specific
sequences.
38
6
FURTHER QUESTIONS ABOUT
EVENTS AT THE ORIGIN(S)
A When DNA replication is complete,
what stops it re-starting until after a
cell has divided?
In eukaryotes,
‘licensing factors’ bind to ORC
and start DNA replicating (p. 33).
Then they are probably degraded,
and only made again
after the cell divides.
39
In E. coli,
active DnaA contains ATP (p. 27).
When DnaA acts,
ATP converts to ADP.
Replacement of ADP by ATP
(i.e. reactivation of DnaA)
is aided by DnaA interacting with the
cell membrane.
Perhaps interaction only occurs when
the membrane is in a particular state,
i.e. perhaps after cell division.
Another possibility in E. coli:
OriC contains DAM sites (p. 26).
Methylation of new DNA at the origin is
delayed.
Perhaps initiation only occurs when the
origin is fully methylated
i.e. perhaps after cell division.
40
B How is initiation at the many origins
of eukaryotes co-ordinated?
Origins are activated in clusters of 20-80,
called ‘replicons’.
‘House-keeping’ genes
(active in most cells)
are in early replicating replicons.
‘Specialised’ genes
replicate early in cells expressing them,
and late in other cells.
Perhaps this pattern allows
genes replicated early to ‘capture’
the available supplies of material
needed for transcription.
41
7 REPLICATION SPEED,
POLYMERISATION SPEED,
FIDELITY AND
THE EVOLUTION OF MULTIPLE
ORIGINS
DNA that is replicating is vulnerable,
so fast replication is needed.
One evolutionary solution is:
fast polymerisation from a single origin
of a small genome.
Bacteria do this.
However, fast polymerisation is,
generally, error-prone.
Another solution is:
slower polymerisation from many origins.
This allows fast replication of,
potentially, high fidelity.
Cells doing this
were also able to develop large genomes,
and evolved into eukaryotes.
42
MB3005
DNA REPLICATION
AIMS:
To consider:
the ‘end problem’:
i.e. how are ends of linear DNA replicated?
Refer to
BI20M3 Lectures on DNA Replication
Lodish, (6th edition, 2008 Chapters 4, 10).
http://www.abdn.ac.uk/~bch118/index.htm
43
1
RECAPITULATION
DNA polymerases catalyse DNA synthesis
in a 5’ to 3’ direction.
They cannot initiate synthesis,
and need a primer with
a 3’ end to extend.
DNA-dependent RNA
(DdRps)
can initiate synthesis.
polymerases
Particular ones (primases) provide
RNA primers for DNA polymerases
during DNA replication.
RNA primers are removed,
e.g. in E. coli by 5’-exonuclease activity of
DNA pol I. [BI20M3]
44
Synthesis of an Okazaki fragment of the
lagging strand:
primase
makes
primer
DNA pol
extends
primer
5’-exo
removes
primer
DNA pol
fills gap
ligase
joins
45
2
THE ‘END PROBLEM’
Problem 1:
Although DdRps (including primases)
do this:
it is not clear that they do this:
46
Problem 2:
Even if they did,
when the primer at the end is removed,
how can it be replaced with DNA?
There is no 3’ end of a preceding
fragment for a DNA polymerase to
extend.
With no replacement,
the products of DNA replication
would look like this:
and each successive round of replication
would shorten the DNA.
47
3
SOME SIMPLE WAYS
SOLVING THE PROBLEM
A
Don’t have linear DNA.
OF
prokaryotes
plasmids
mitochondria
chloroplasts
many viruses
carry genetic material as
closed, circular DNA molecules.
B
Convert linear DNA to circular
DNA for replication.
E.g.
phage  DNA is linear in the virion,
but circularised in the infected cell.
48
4
TELOMERES
Another method is to counteract the
predicted shortening of the DNA ends
by using an enzyme to extend them.
This occurs in eukaryotic cells.
The ends of linear eukaryotic DNA are
called telomeres.
A telomere 3’ end
consists of short ‘G-rich’ sequences
tandemly repeated many times.
Early work on these used Tetrahymena,
a protozoon containing many DNA
fragments,
and hence telomeres.
Here, the repeat sequence is TTGGGG.
In man, it is TTAGGG.
49
A telomere 5’ end has complementary,
C-rich repeats,
and the 3’ end has a 12- to 16-mer
overhang.
In summary,
a telomere looks like this:
50
When DNA is not replicating,
the G-rich overhang
folds back on itself,
to protect chromosome ends
from nucleases
and recombination with other DNA.
Probably,
stacked, H-bonded, 4-stranded
‘G-quartets’
form.
51
When DNA replicates,
shortening of the ends occurs
(as predicted in Problem 2).
If it continued,
after several replication rounds,
information-carrying DNA
would begin to be lost.
But loss of G-rich repeats
is counteracted by an enzyme
that adds them:
telomerase.
52
5
TELOMERASE
Telomerase is a ribonucleoprotein.
Its single RNA molecule has a sequence,
near its 5’ end,
complementary to the G-rich repeat at
the 3’ end of the telomere.
So,
telomerase is a reverse transcriptase,
that carries with it its own template.
53
G-rich repeats may be made by an
‘inchworm’ mechanism:
5’ end of
telomerase
RNA
3’ end of
telomere
RNA acts
as template
telomerase RNA
inches along to act
as template again
54
Then,
the G-rich 3’ end sequence
is used as a template to lengthen
the C-rich 5’ end of the telomere.
Telomere lengthening is controlled:
telomere-binding proteins limit access of
telomerase to telomeres;
and, conversely,
a protein, ‘tankyrase’,
covalently modifies
telomere-binding protein(s),
and removes them from the telomere,
allowing telomerase access.
55
6 EVOLUTION OF THE TELOMERE/
TELOMERASE SYSTEM
Telomerase protein is similar in sequence
to retrotransposon reverse transcriptases:
Drosophila
telomeres
are
unusual,
consisting of repeats of two known
retrotransposons.
Perhaps other telomeres are degraded
retrotransposons.
56
7
TELOMERASE, AGEING AND
CANCER
Human somatic cells:

express no/low telomerase activity;

in the body and in culture,
gradually lose telomere sequences,
and eventually die;

in people with progeria
(genetic trait causing premature
ageing and death in childhood)
have very short telomeres;

in people with dyskeratosis congenita
(genetic trait causing problems in
rapidly turned-over tissue:
skin, nails, hair, gut, bone marrow)
have mutations in the gene
encoding telomerase RNA.
57
Gametes,
most cancer cells,
unicellular eukaryotes:

express telomerase;

maintain telomere length through
indefinite numbers of cell divisions.
Could telomerase
ageing?
restoration
slow
Could telomerase
cancers?
inhibition
stop
58
Dolly’s telomeres
Dolly was cloned from a cell of an adult
sheep.
Would her ‘telomere clock’ be set back
to that of a newly born lamb?
In fact, her telomeres were much shorter
than expected for her age,
putting in doubt potential treatments
involving implanting (adult) cells in
patients with
e.g.
liver failure,
Parkinson’s disease.
However, subsequently cloned calves
have longer telomeres than expected.
59
Some easily accessible (electronic journal) review
articles for further reading
1
SUPERCOILING
Corbett, K.D. & Berger, J.M. (2004) Structure, molecular mechanisms, and
evolutionary relationships in DNA topoisomerases. Annual Review of Biophysics
and Biomolecular Structure 33 95-118.
http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.biophys.33.110502.140357;jsessionid=iwBAXcm_mcdc_5-qaP
Schvartzman, J.B. & Stasiak, A. (2004) A topological view of the replicon.
EMBO Reports 5, 256-261.
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1299012&blobtype=pdf
2
ORIGINS
Cvetic, C. & Walter, J.C. (2005) Eukaryotic origins of DNA replication: could
you please be more specific? Seminars in Cell & Developmental Biology 16 343353.
http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WX0-4FJV20S-31&_cdi=7144&_user=152381&_orig=search&_coverDate=06%2F30%2F2005&_qd=1&_sk=999839996&view=c&wchp=dGL
bVlb-zSkzS&md5=ced07478c4d7577ce9a308b4a0fd335d&ie=/sdarticle.pdf
Robinson, N.P. & Bell, S.D. (2005) Origins of DNA replication in the three
domains of life. FEBS Journal 272 3757-3766.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16045748&query_hl=10
&itool=pubmed_DocSum
3
‘END PROBLEM’
Blackburn, E.H. (2005) Telomeres and telomerase: their mechanisms of action
and the effects of altering their functions. FEBS Letters 579 859-862.
http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T36-4DWGS1N-71&_cdi=4938&_user=152381&_orig=search&_coverDate=02%2F07%2F2005&_qd=1&_sk=994209995&view=c&wchp=dGL
bVtz-zSkzk&md5=47fc6f654a8673e2cffa8b4bf7c3562a&ie=/sdarticle.pdf
Shin, J.-S., Hong, A., Solomon, M.J. & Lee, C.S. (2006) The role of telomeres and
telomerase in the pathology of human cancer and aging. Pathology 38, 103-113.
http://taylorandfrancis.metapress.com/media/788y6huvqpdwtcu2qrrq/contributions/q/0/7/2/q07245t610216rh8.pdf