ADN Replication

Chapter 11 : DNA Replication
Outline:
* Semiconservative Replication
o Meselson-Stahl Experiment
* DNA polymerases and DNA elongation
* Molecular model of DNA replication
o Initiation of Replication
o Semidiscontinuous DNA replication
o Rolling circle replication
* Replication of telomeres in eukaryotes
DNA replication underlies the process of inheritance at all levels (cellular, organismal,
population). DNA replication occurs as prelude to cell division ( S phase of cell cycle in
eukaryotes). DNA in all organisms is the end point in a continuous series of replications
going back to the origin of life, almost 4 billion yrs ago. DNA replication is based on
complementarity of DNA molecules and on ability of proteins to form specific interactions
with specific sequences of DNA.
Semiconservative Replication
* Watson and Crick model of DNA suggested that each strand could serve as template for
synthesis of new strand. Their model is called semiconservative DNA replication
* Two other models based on template-based synthesis were also proposed by others
(Fig 11.1):
o Conservative model: parental strands rejoin after they are used as templates, resulting
in two DNA moleucles, one made of two parental strands, and the other made entirely of
newly synthesized DNA.
o Dispersive model: parental DNA cleaved into DNA segments that act as templates for
the synthesis of new DNA and then somehow segments reassemble into double stranded
DNA made of parental and progeny DNA which are interspersed.
o All three models made different predictions about the nature of DNA after one and two
rounds of replication (Fig 11.1).
Meselson-Stahl Experiment
* Meselson and Stahl (1958) used a heavy isotope of nitrogen (15N) and equilibrium

density gradient centrifucation to show that DNA replicated in semiconservative manner
in E. coli (Fig11.2).
o grew E. coli for many generations in medium containing 15NH4Cl (15N is a heavier
isotope than 14N). This resulted in DNA containing 15N instead of 14N. 15N DNA can be
seperated from 14N DNA by ultracentifugation in a CsCl gradient.
o 15N-labeled bacteria were then transferred to medium containing 14N and allowed to
grow for several generations, and sampled after each replication cycle.
o After one generation in 14N, all the DNA had a density intermediate from 15N-DNA and
14N-DNA, just as predicted by the semiconservative and dispersive models.
+ this result ruled out the conservative model because it predicted that there should be
two bands (one containing light DNA and the other heavy DNA).
o To distinguish between the semiconservative model and the dispersive model, E.coli
were grown for another generation. Two bands were observed, as expected by the
semiconservative model. The dispersive model predicted that there should only be one
band, therefore it was also ruled out. The results were all consistent with the
semiconservative model.
o
* Semiconservative DNA replication also occurs in eukaryotes (see harlequin
chromosomes in Fig 11.3).
DNA polymerases and DNA elongation
* In 1955, Arthur Korberg identified the first DNA polymerase (DNA Pol I). Initially it was
thought to be the main DNA replication enzyme, but mutant E.coli defective in the gene
encoding for DNA pol I divided normally, indicating that there must be other enzymes
involved.
* Five DNA polymerases have now been identified in E. coli. DNA Pol II, IV, and V are
involved in DNA repair. DNA pol I and III are involved in DNA replication.
* All DNA polymerases catalyze the polymerization of nucleotide precursors (dNTPs) into
a DNA chain . The reaction is shown in Fig 11.4 and has three main features:
1. DNA pols catalyze the formation of a phosphodiester bond between the 3'-OH group of
the deoxyribose on the last nucleotide in the chain and the 5'-phosphate of the incoming

nucleotide. The energy is supplied by the hydrolysis of the two phosphates from the
dNTP. All DNA polymerases require a primer (i.e they can not add the first nucleotide ).
2. DNA polymerases require a template. The particular nucleotide added depends on
correct complementary base pairing with the template. DNA pols are fast. In E. coli, DNA
pol I and II can polymerize ~ 850 nt per sec. In humans, its a lot slower (60-90 nt/sec).
3. All DNA polymerases synthesize DNA in the 5' to 3' direction.
* DNA pol I and II also have exonuclease activity.
o DNA pol I and III have 3'-> 5' exonuclease activity. This is a proofreading mechanism.
DNA pols add an incorrect base with a frequency of 10-6. When an incorrect base is
added, the enzyme detects that it made a mistake, and uses its 3' to 5' exonuclease
activity to move back and remove the incorrect base. With proofreading, the error rate
drops to 10-9.
o DNA pol I also has 5' -> 3' exonuclease activity. This allows it to remove DNA or RNA
from the 5' end of a moleecule. This is essential during DNA replication of the lagging
strand.
Model of DNA Replication in E. coli
* The bare-bone mechanics of DNA replication is similar in all organisms. However, we
will only focus on DNA replication in E. coli, where it is best understood. Along the way,
significant differences between prokaryotic and eukaryotic DNA replication will be
highlighted.
* Basic research into the mechanisms of DNA replication in E. coli (as well as
transcription and translation) has led to the identification and cloning of dozens of genes
involved in these processes (Table 11.1). The creative use of these gene products has
given us a tremendous power to manipulate genes and genomes according to our will.
Initiation of Replication
* Initiation of replication starts at a DNA sequence called the replicator, which includes
the origin of replication (OriC) (AT-rich) where DNA is denatured into single strands to
form a replication bubble. At either end of a bubble there is a replication fork, where DNA
synthesis occurs, using each separated strand as a template.
o Circular genomes of prokaryotes contain a single origin of replication.

o In eukaryotes, linear chromosomes contain many origins of replication (allows faster
replication).
o Synthesis proceeds bidirectionaly at replication fork. Eventually, replicated double
helices join each other, producing two daughter molecules (Fig 11.9)(sister chromatids,
in eukaryotes)
* Initiation of replication starts with the binding of an initiator protein which denatures
the oriC and then recruits a DNA helicase (one for each strand) which untwists the DNA
in both directions (energy comes from hydrolysis of ATP) (Fig 11.5).
* Next, each helicase recruits a DNA primase to form a primosome. DNA primase makes
the necessary RNA primers ( 5-10 nts) needed by DNA polymerase III.
* The next step involves the assembly of the rest of the proteins involved in DNA
replication. These proteins associate to form a replisome. There is a replisome at each
replication fork.
Semidiscontinuous DNA replication
* The replication steps are identical at each replication fork, so we focus on just one. The
entire process is shown in Fig 11.6.
* After the helicase unwinds the DNA, the single stranded DNA is prevented from
reannealing by binding to single-strand DNA-binding proteins (SSBs) (about 200 /rep
fork).
* DNA pol III dimer (part of replisome) now initiates polymerization by adding dNTPs to
the RNA primer on each of the strands. Because strands in double helix are in antiparallel
configuration, and DNA polymerases add dNTPs in 5' to 3' direction, the two strands are
synthesized differently:
o Leading strand synthesized continuously; only one primer required; DNA pol III moves
in same direction as replication fork.
o Lagging strand synthesized discontinuously as Okazaki fragments, which are later
ligated by DNA ligase. Each Okazaki fragment requires a primer. DNA pol III moves in
opposite direction to replication fork.
* In Lagging-strand synthesis, DNA Pol III ends polymerization when it encounters
double stranded DNA ahead (from previous Okazaki fragment). It dissociates from the

DNA, leaving a gap in one strand. This gap is recognized as damaged DNA and is
repaired by DNA Pol I.
* DNA Pol I removes primers and fills in gaps (has 5'-3' exonuclease activity).
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* DNA ligase joins 3' end of one Okazaki fragment to 5' end of downstream Okazaki
fragment (Fig 11.7).
* As helicase unwinds DNA ahead of replication fork, positive supercoils form elsewhere
in the molecule. For replication fork to move, the helix must rotate (estimated at 50
revolutions/sec). The problem of supercoiling is solved by the action of topoisomerases
(specifically a Gyrase) which introduce negative supercoils to counteract positive
supercoils intoduce by helicases.
Rolling circle replication
* For many viral DNAs and some plasmids (e.g. F plasmid in E. coli), rolling circle
replication has been demonstrated.
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* Synthesis usually continues beyond a single chromosomal unit. This results in many
head-to-tail copies of the plasmid, which is then cut and rejoined into new circular
molecules.
Replication of telomeres in eukaryotes
* There are special problems associated with replication of the ends of linear
chromosomes (called telomeres). Recall that DNA polymerases only add nucleotides to
the 3' end of a growing chain. When the linear chromosomes of eukaryotes replicate, the
resulting daughter molecules will each have an RNA primer left over at the 5'end (Fig
11.14). This RNA primer is removed, leaving a single stranded DNA segment. If not fixed,
this single-stranded DNA region will get degraded, and the linear chromosomes will get
shorter with each round of DNA replication.
* In most eukaryotes, an enzyme called telomerase, maintains the ends of chromosomes
by adding telomere repeats to chromosome ends. The mechanism is shown below (and in
Fig 11.5).
* Telomerase is a ribonucleoprotein (has RNA molecule as part of its structure) which

adds tandem repeats to the 3' end of chromosomes using an RNA molecule as a
template. After is has added many telomeric repeats and has left, a new DNA molecule is
made starting from a new RNA primer, which is again is removed, but by this time the
chromosme has already been extended.
* The absence of telomerase activity in cells is correlated with senescence of cells (i.e.
die after certain number of cell divisions). Conversely, enhanced telomerase activity
correlated with cell immortalily (i.e. cells divide indefinately).
o cells with short telomerse undergo fewer doublings than ones with long telomerase.
o fibroblasts form individuals with progeria (rare disease characterized by premature
aging) have short telomeres.
o most somatic cells have no active telomerase (divide only 20-60 times)
o sperm cells, stem cells and unicellular eukaryotes (essentially immortal ) have active
telomerase and stable telomeres.
o cancer cells, which are also essentially immortal, have active telomerase (promising
target for drug design)
o Elimination of telomerase activity in somatic cells may be a cellular senescence
mechanism that protects multicellular organisms from cancer
Chapter 13 : Transcription
* Outline
o Genes and RNA
o Properties of RNA
o Classes of RNA
o Making functional transcripts
+ RNA polymerases
+ Initiation
+ Elongation
+ Termination
o RNA processing in eukaryotes
Genes and RNA
Biological information flow from DNA to protein requires an RNA intermediate. RNA is

produced by a process that copies the nucleotide sequence in DNA to produce a
transcript. This process is called transcription.
Properties of RNA
1. Single stranded, but can undergo intramolecular base-pairing
- forms variety of 3D structures specified by sequence.
2. Ribose sugar (not deoxyribose)
3. Uracyl in place of thymine
Classes of RNA
* There are a variety of different RNAs that can be classified into two classes.
o 1. Informational RNAs (e.g. messenger RNA)
+ intermediate which is later translated into protein.
+ most genes encode mRNA
o 2. Functional RNAs
+ never translated
+ diverse roles in cell
+ main classes of functional RNAs play critical roles in various steps in the information
processing of DNA to protein:
# rRNA - components of ribosome
# tRNA - bring amino acids to mRNA during translation
# snRNA (small nucleolar RNAs) - involved in splicing of introns
# scRNAs (small cytoplasmic RNAs) - protein trafficking
* All DNA and RNA function is based on two key elements:
o 1. Complementary bases in single stranded nucleotide chains can H-bond to form
double stranded structures.
o 2. Specific sequences can be recognized by specific nucleic-acid binding proteins.
Making functional transcripts
* Transcription uses one DNA strand as template
o Strands of double helix must be separated, so that one of these strands (template
strand) can serve as template to direct the synthesis of transcript.
* Either strand along the chromosome can serve as template, but for a given gene, its

always the same strand.
* RNA polymerase catalyzes the synthesis of RNA using DNA template (Fig 13.1).
o RNA grows in 5' to 3' direction, and the template is read in the 3' to 5' direction.
o sequence of RNA is complementary to template strand (noncoding strand), but the
same as nontemplate strand (coding strand) except T replaced with U.
* A typical prokaryotic gene has the folowing features:
RNA Polymerases
* Prokaryotes have only one RNA Polymerase but eukaryotes have 3:
1. RNA Pol I: transcribes rRNA genes
2. RNA Pol II: transcribes protein encoding genes
3. RNA Pol III: transcribes other functional RNAs (tRNAs, snoRNAs etc...)
* In eukaryotes, transription takes place in nucleus.
* In prokaryotes, transcription and translation are coupled.
* Transcription involves 3 distinct stages: initiation, elongation, and termination.
Initiation
* In E. coli, transcription requires a complex of RNA polymerase and the sigma factor (s)
which binds to a promoter. The RNA polymerase core enzyme (4 has four subunits, two
a, one b and one b') complexed with the sigma factor is known as the holoenzyme. Once
transcription is initiated, the sigma factor dissociates.
* promoter = DNA sequence to which RNA Pol binds to initiate transcription.
o note that by convention, gene is labelled the same way as RNA transcript. So promoter
is at 5' end of gene (Fig 13.3).
* RNA pol + sigma factor scans DNA for promoter sequence, binds DNA at the promoter
sequence (- 10 region and -35 region), unwinds it, and begins synthesis of a transcript at
transcription initiation site. Promoter sequences are not transcribed. NOTE: RNA pol does
not need a primer to initiate RNA synthesis not does it need a helicase.
o there are consensus sequences for all promoters in E. coli. A consensus sequence is the
sequence found most frequently at each position. E.g consensus sequence at -10 position
is 5'-TATAAT-3'
o The more similar the promoter sequence is to the consensus, the higher the rate of

transcription.
o It is the sigma factor that binds the promoter. Different sigma factors bind different
promoters.
* What is described above is the minimum required for transcription initiation. In chapter
19 we will study how genes are regulated in prokaryotes in more detail.
Elongation
* RNA pol moves along DNA, maintaining transcription "bubble" to expose template
strand, and catalyzes the 3' elongation of transcript.
o energy for reaction derived from splitting high-energy triphosphates into
monophosphates.
o rate of transcription is about 30-50 nt/sec
Termination
* Results from different mechanisms signalled by termination sequences at 3' end of a
gene. Two mechanisms known:
o Rho-independent termination
+ involves formation of hairpin loop (Fig 13.5) in nascent transcript causing RNA strand
and RNA Pol to be released from DNA template.
o Rho-dependent termination