José A. Cardé- Serrano, PhDUniversidad Adventista de las AntillasBiol 223 – GenéticaAgosto 2010

Basic Features of DNA Replication In Vivo

DNA Polymerases and DNA Synthesis In Vitro

The Complex Replication ApparatusUnique Aspects of Eukaryotic

Chromosome replication

Replicación – procesos por el cual la célula genera una copia de su material genético usando como molde una previa

Iniciación extensión terminación

30,000 bpm vs 3,000 bpm1 error / billón Implicaciones en los gemelos?

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 in a hybrid new molecule

Problema: Pregunta? Como se replicará el DNA?

3 Hipótesis posibles

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

Mg2+

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' 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 phaseMultiple origins of replicationNucleosomesTelomeres

DNA polymerase -DNA primase—initiation; priming of Okazaki fragments

DNA polymerase —processive DNA synthesis

DNA polymerase —DNA 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 (and/or ) 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.