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Jos A. Card- Serrano, PhD
Universidad Adventista de las
Antillas
Biol 223 Gentica
Agosto 2010
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Basic Features of DNA Replication In Vivo DNA Polymerases and DNA Synthesis In Vitro
The Complex Replication Apparatus Unique Aspects of Eukaryotic Chromosome
replication
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DNA replication occurs semiconservatively, is initiated at unique origins,and usually proceeds bidirectionally from each origin of replication.
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Each strand serves asa template
Complementary basepairing determinesthe sequence of thenew strand
Each strand of theparental helix isconserved
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DNA replicates by a semiconservative mechanism: as thetwo complementary strands of a parental double helixunwind and separate, each serves as a template for thesynthesis of a new complementary strand.
The hydrogen-bonding potentials of the bases in thetemplate strands specify complementary base sequences inthe nascent DNA strands.
Replication is initiated at unique origins and usually proceedsbidirectionally from each origin.
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Much of what we know about DNA synthesis was deduced from in vitrostudies.
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Primer DNA with free3'-OH
Template DNA tospecify the sequenceof the new strand
Substrates: dNTPs
Mg2+
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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 and/or
Replication of Mitochondrial DNA: Polymerase
DNA Repair: Polymerases and
All of these enzymes synthesize DNA 5' to 3' andrequire a free 3'-OH at the end of a primer
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DNA synthesis is catalyzed by enzymes called DNApolymerases.
All DNA polymerases require a primer strand, which isextended, and a template strand, which is copied.
All DNA polymerases have an absolute requirement for afree 3'-OH on the primer strand, and all DNA synthesis
occurs in the 5' to 3' direction.
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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.
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DNA replication is a complex process, requiring the concerted action of alarge number of proteins.
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Synthesis of the leading strand is
continuous.
Synthesis of the lagging strand isdiscontinuous. The new DNA is synthesized
in short segments (Okazaki fragment) that
are later joined together.
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Okazaki Fragments Experiment
- Crecer fagos y Ecoli en medio con3H Timina por periodos cortos (pulso
y seguimiento)
- Aislar el DNA y centrifugarlos paramedir 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
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DNA replication is complex, requiring the
participation of a large number of proteins.
DNA synthesis is continuous on the progeny strandthat is being extended in the overall 5'3' direction,but is discontinuous on the strand growing in the
overall 3'5' direction.
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New DNA chains are initiated by short RNA primers
synthesized by DNA primase.
The enzymes and DNA-binding proteins involved inreplication assembled into a replisome at eachreplication fork and act in concert as the fork moves
along the parental DNA molecule.
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Although the main features of DNA replication are the same in allorganisms, some processes occur only in eukaryotes.
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Shorter RNA primers and Okazaki fragments DNA replication only during S phase
Multiple origins of replication Nucleosomes Telomeres
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DNA polymerase -DNAprimaseinitiation;priming of Okazaki
fragments DNA polymerase
processive DNA synthesis
DNA polymerase DNA
replication and repair invivo
PCNA (proliferating cellnuclear antigen)slidingclamp
Replication factor-C Rf-C)
loading of PCNA Ribonuclease H1 and
Ribonuclease FEN-1
removal of RNA primers
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Most human somaticcells lack telomeraseactivity.
Shorter telomeres areassociated with cellularsenescence and death.
Diseases causing
premature aging areassociated with shorttelomeres.
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The large DNA molecules in eukaryotic chromosomereplicate bidirectionally from multiple origins.
Two or three DNA polymerases (and/or ) are present at
each replication fork in eukaryotes.
Telomeres, the unique sequences at the ends ofchromosomes, are added to chromosome by a uniqueenzyme called telomerase.