Jos A.

Card- Serrano, PhD

Universidad Adventista de las
Antillas
Biol 223 Gentica
Agosto 2010
Basic Features of DNA Replication In Vivo
DNA Polymerases and DNA Synthesis In Vitro
The Complex Replication Apparatus
Unique Aspects of Eukaryotic Chromosome
replication
DNA replication occurs semiconservatively, is initiated at unique origins,
and usually proceeds bidirectionally from each origin of replication.
Each strand serves as
a template
Complementary base
pairing determines
the sequence of the
new strand
Each strand of the
parental helix is
conserved
DNA replicates by a semiconservative mechanism: as the
two complementary strands of a parental double helix
unwind and separate, each serves as a template for the
synthesis of a new complementary strand.

The hydrogen-bonding potentials of the bases in the
template strands specify complementary base sequences in
the nascent DNA strands.

Replication is initiated at unique origins and usually proceeds
bidirectionally from each origin.
Much of what we know about DNA synthesis was deduced from in vitro
studies.
Primer DNA with free
3'-OH
Template DNA to
specify the sequence
of the new strand
Substrates: dNTPs
Mg
2+

Polymerases in E. coli
DNA Replication: DNA Polymerases III and I
DNA Repair: DNA Polymerases II, IV, and V
Polymerases in Eukaryotes
Replication of Nuclear DNA: Polymerase o, o and/or c
Replication of Mitochondrial DNA: Polymerase
DNA Repair: Polymerases |, c, k, ,, , and q
All of these enzymes synthesize DNA 5' to 3' and
require a free 3'-OH at the end of a primer
DNA synthesis is catalyzed by enzymes called DNA
polymerases.

All DNA polymerases require a primer strand, which is
extended, and a template strand, which is copied.

All DNA polymerases have an absolute requirement for a
free 3'-OH on the primer strand, and all DNA synthesis
occurs in the 5' to 3' direction.
The 3'5' exonuclease activities of DNA
polymerases proofread nascent strands as they are
synthesized, removing any mispaired nucleotides at
the 3' termini of primer strands.
DNA replication is a complex process, requiring the concerted action of a
large number of proteins.
Synthesis of the leading strand is
continuous.
Synthesis of the lagging strand is
discontinuous. The new DNA is synthesized
in short segments (Okazaki fragment) that
are later joined together.
Okazaki Fragments Experiment
- Crecer fagos y Ecoli en medio con
3H Timina por periodos cortos (pulso
y seguimiento)

- Aislar el DNA y centrifugarlos para
medir su grado de Sedimentacion

-A periodos cortos de 5, 10, 15, 20
segundos se obtienen fragmentos
bien cortos

- A periodos mas largos, la
radioactividad se ve incluida en
fragmentos mas grandes

DNA replication is complex, requiring the
participation of a large number of proteins.

DNA synthesis is continuous on the progeny strand
that is being extended in the overall 5'3' direction,
but is discontinuous on the strand growing in the
overall 3'5' direction.
New DNA chains are initiated by short RNA primers
synthesized by DNA primase.

The enzymes and DNA-binding proteins involved in
replication assembled into a replisome at each
replication fork and act in concert as the fork moves
along the parental DNA molecule.
Although the main features of DNA replication are the same in all
organisms, some processes occur only in eukaryotes.
Shorter RNA primers and Okazaki fragments
DNA replication only during S phase
Multiple origins of replication
Nucleosomes
Telomeres
DNA polymerase o-DNA
primaseinitiation;
priming of Okazaki
fragments
DNA polymerase o
processive DNA synthesis
DNA polymerase cDNA
replication and repair in
vivo
PCNA (proliferating cell
nuclear antigen)sliding
clamp
Replication factor-C Rf-C)
loading of PCNA
Ribonuclease H1 and
Ribonuclease FEN-1
removal of RNA primers
Most human somatic
cells lack telomerase
activity.
Shorter telomeres are
associated with cellular
senescence and death.
Diseases causing
premature aging are
associated with short
telomeres.
The large DNA molecules in eukaryotic chromosome
replicate bidirectionally from multiple origins.

Two or three DNA polymerases (o, o, and/or c) are present at
each replication fork in eukaryotes.

Telomeres, the unique sequences at the ends of
chromosomes, are added to chromosome by a unique
enzyme called telomerase.