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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.

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