VIII. DNA Function: Replication

VIII. DNA Function: Replication

DNA Function: Replication
A. Recap - occurs in the S-phase of Interphase - unreplicated chromosomes, each consisting of a complementary double-helix, is REPLICATED: produce a chromosomes with 2 identical chromatids. - Once DNA replication has occurred, cells will proceed to division.

A. Recap - occurs in the S-phase of Interphase - unreplicated chromosomes, each consisting of a complementary double-helix, is REPLICATED: produce a chromosomes with 2 identical chromatids. - Once DNA replication has occurred, cells will proceed to division. B. Hypotheses:

A. Recap - occurs in the S-phase of Interphase - unreplicated chromosomes, each consisting of a complementary double-helix, is REPLICATED: produce a chromosomes with 2 identical chromatids. - Once DNA replication has occurred, cells will proceed to division. B. Hypotheses: 1. Conservative Model: The original DNA is used as a template for the formation of new strand, and then they reanneal: Reannealing of original strands and synthesis of new double helix. new strand formation Original DNA

A. Recap - occurs in the S-phase of Interphase - unreplicated chromosomes, each consisting of a complementary double-helix, is REPLICATED: produce a chromosomes with 2 identical chromatids. - Once DNA replication has occurred, cells will proceed to division. B. Hypotheses: 1. Conservative Model: The original DNA is used as a template for the formation of new strand, and then they reanneal : Reannealing of original strands and synthesis of new double helix. new strand formation Original DNA

A. Recap - occurs in the S-phase of Interphase - unreplicated chromosomes, each consisting of a complementary double-helix, is REPLICATED: produce a chromosomes with 2 identical chromatids. - Once DNA replication has occurred, cells will proceed to division. B. Hypotheses: 1. Conservative Model: 2. Semi-conservative Model: The original DNA is used as a template for the formation of new strand, which then bind together: Binding of old and new strands new strand formation Original DNA

A. Recap - occurs in the S-phase of Interphase - unreplicated chromosomes, each consisting of a complementary double-helix, is REPLICATED: produce a chromosomes with 2 identical chromatids. - Once DNA replication has occurred, cells will proceed to division. B. Hypotheses: 1. Conservative Model: 2. Semi-conservative Model: 3. Dispersive: Old and new DNA distributed throughout double-helix Dispersal of old and new DNA throughout both chromatids new strand formation Original DNA

A. Recap B. Hypotheses C. The Meselson and Stahl Experiment (1958)

C. The Meselson and Stahl Experiment (1958)
- They grew E. coli for many generations on an agar where the available Nitrogen was a heavy isotope. The heavy nitrogen was incorporated into NEW DNA that is synthesized by the cell.

- They grew E. coli for many generations on an agar where the available Nitrogen was a heavy isotope. The heavy nitrogen was incorporated into NEW DNA that is synthesized by the cell. When the DNA is spun in a centrifuge, it spins down ‘far’

- Then, grew bacteria on an N15 medium for 1 generation and took samples. What banding pattern would you expect to see under each of the three hypotheses?

- Then, grew bacteria on an N15 medium for 1 generation and took samples. What banding pattern would you expect to see under each of the three hypotheses? - Results refuted the conservative hypothesis.

- Then, grew bacteria on an N15 medium for 1 generation and took samples. What banding pattern would you expect to see under each of the three hypotheses? Results refuted the conservative hypothesis. Grew bacteria for a second generation on N14 medium…. Predictions of 2 models?

- Then, grew bacteria on an N15 medium for 1 generation and took samples. What banding pattern would you expect to see under each of the three hypotheses? Results refuted the conservative hypothesis. Grew bacteria for a second generation on N14 medium…. Predictions of 2 models? Results refuted the dispersive model and confirmed the semi-conservative model.

D. Replication at the Molecular Level

1. Replication in E. coli a. A specific sequence of bases is recognized as the binding site for the replication complex – this is called the Replication Origin, and the DNA replicated from this site is called a replicon.

1. Replication in E. coli a. A specific sequence of bases is recognized as the binding site for the replication complex – this is called the Replication Origin, and the DNA replicated from this site is called a replicon. - Bacteria have only a single replication origin, and the entire circular chromosome is replicated from this point.

1. Replication in E. coli a. Replication Origin has 9-mers and 13-mers – homologous sequences… b. Three enzymes (DNaA,B, C), collectively called “helicases” bind to the origin and break the hydrogen bonds holding the helices together. “Single-strand binding proteins” stabilize the DNA.

1. Replication in E. coli a. Replication Origin has 9-mers and 13-mers – homologous sequences… b. Three enzymes (DNaA,B, C), collectively called “helicases” bind to the origin and break the hydrogen bonds holding the helices together. “Single-strand binding proteins” stabilize the DNA. c. The enzyme gyrase works downstream, cutting single or double strands to relieved the torque on the molecule. (topoisomerases affect shape).

1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I.

1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I. DNA Poly III is the primary replication enzyme, but I-V have repair functions

1. Replication in E. coli d. DNA Polymerases: And note that NONE can initiate chain synthesis!!!!

1. Replication in E. coli d. DNA Polymerases: There are 5 enzymes that can lengthen a DNA strand by adding new bases. The first was isolated in 1957 by A. Kornberg (1959 Nobel) – DNA Polymerase I. DNA Poly III is the primary replication enzyme, but I-V have repair functions DNA Poly’s are very complex enzymes with multiple polypeptides and functional groups. And actually, two DNA Poly III enzymes work in parallel as a DIMER

1. Replication in E. coli d. DNA Polymerases: The DNA Polymerases, the helicases and Gyrase, the Stabilization Proteins, and other enzymes we’ll mention form a HUGE functional replication unit = Replisome

D. DNA Replication at the Molecular Level 1. Replication in E. coli
2. The Process – Replication at the fork: a. Initiation: i . Helicases bind and separate helices, stabilizing protein stabilize the single strands, gyrase relives torque ii. Primase, an RNA Polymerase, begins synthesis of an RNA strand in the 5’3’ direction on both strands: 3’ 5’ 3’

2. The Process – Replication at the fork:
a. Initiation: b. Polymerization: NOW, with a free-3’-OH to add bases to, DNA Polymerase III displaces the Primase and adds DNA bases to extend the new helix in both directions bases/min!):

2. The Process – Replication at the fork:
a. Initiation: b. Polymerization: the B-subunit of DNA Poly III “is a “sliding clamp” that holds the enzymes on the helices:

videos Video 2

2. The Process a. Initiation: b. Polymerization: As the replisome moves down the helix, replication is continuous on the “leading strand”, 5’3’, and discontinuous on the “lagging strand” (though the new strand is still synthesized 5’3’).

2. The Process a. Initiation: b. Polymerization: As the replisome moves down the helix, replication is continuous on the “leading strand”, 5’3’, and discontinuous on the “lagging strand” (though the new strand is still synthesized 5’3’). The short sections of discontinuously synthesized DNA are called “Okazaki Fragments”.

2. The Process a. Initiation: b. Polymerization: As the replisome moves down the helix, replication is continuous on the “leading strand”, 5’3’, and discontinuous on the “lagging strand” (though the new strand is still synthesized 5’3’). The short sections of discontinuously synthesized DNA are called “Okazaki Fragments”. This is what is happening at one fork; how about the other?

5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: The RNA must be removed… 3’ 5’

5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: DNA Poly I, with 5’3’ exonuclease capability, cuts out the RNA primer… 3’ 5’ 3’ 5’

5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: DNA Poly I, with 5’3’ exonuclease capability, cuts out the RNA primer…DNA Poly I and II, “fill the gap” with DNA bases, using the free-3’-OH group on the last DNA base to add bases to.... the epsilon subunit is critical in repair… 3’ 5’ 3’ 5’

5’ 3’ 2. The Process a. Initiation: b. Polymerization: c. Repair: DNA Poly I, with 5’3’ exonuclease capability, cuts out the RNA primer…DNA Poly I and II, “fill the gap” with DNA bases, using the free-3’-OH group on the last DNA base to add bases to… then Ligase make the last phosphodiester bond, linking the fragments of DNA together. 3’ 5’

2. The Process a. Initiation: b. Polymerization: c. Repair: d. Termination: With a circular chromosome (bacteria), it is easy to complete the process.

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication.

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication. - mammalian cells have 25,000 replicons; in eukaryotes these are Autonomously Replicating Sequences (ARS).

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication. - mammalian cells have 25,000 replicons; in eukaryotes these are Autonomously Replicating Sequences (ARS). - How are they found by the replisome? - proteins bind to these specific sequences/regions during the G1 phase – these proteins form an “Origin Recognition Complex” (ORC).

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins - with 103 – 106 times as many bases, multiple origins to speed the replication process by simultaneous replicon replication. - mammalian cells have 25,000 replicons; in eukaryotes these are Autonomously Replicating Sequences (ARS). - How are they found by the replisome? - proteins bind to these specific sequences/regions during the G1 phase – these proteins form an “Origin Recognition Complex” (ORC). - at the onset of the S phase, protein kinase enzymes bind and form a ‘pre-replication complex’ that binds the DNA Polymerase and helicases and initiates replication. The activated kinases, bound with the replisome, inhibit the formation of more pre-RC, so replication of a region occurs only once.

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins b. Histone Complexes: - The ‘core’ DNA bound to histones in a nucleosome must be unwound to be replicated. After this occurs, new histones are made to bind the new double-helices.

1. Replication in E. coli 2. The Process - Replication at the fork 3. Modifications in Eukaryotes a. Multiple Origins b. Histone Complexes: c. Polymerase Production - base replication rates are 25x slower in eukaryotes - but they have multiple origins and 50,000 x as many Polymerase III enzymes…so the process occurs faster at a cellular level.

3. Modifications in Eukaryotes
a. Multiple Origins b. Histone Complexes: c. Polymerase Production d. Telomere Replication - Problem: shortening chromosomes RNA primer

3. Modifications in Eukaryotes
a. Multiple Origins b. Histone Complexes: c. Polymerase Production d. Telomere Replication - Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations).

d. Telomere Replication
- Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations). - Telomerase is a ribonucleoprotein, with an enzymatic RNA molecule coupled to a protein.

- Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations). - Telomerase is a ribonucleoprotein, with an enzymatic RNA molecule coupled to a protein. The RNA has several repeats of CCCCAA, which act as a template for the formation of GGGGTT DNA in the process of reverse transcription (reading RNA and making DNA).

- Problem: shortening chromosomes - This is bad, because intact telomeres protect the chromosomes from degradation and end-end fusion (translocations). - Telomerase is a ribonucleoprotein, with an enzymatic RNA molecule coupled to a protein. The RNA has several repeats of AACCCC, which act as a template for the formation of TTGGGG DNA in the process of reverse transcription (reading RNA and making DNA). The process continues, forming a long and non-coding platform for primase to begin discontinuous synthesis.

- Problem: shortening chromosomes - Curiously, most mature cells lack telomerase. The shortening of chromosomes may be a signal, or an inhibitor, that prevents further cell division. GOOD because mutations accumulate in a lineage with each division BAD because it reduces rate of tissue repair = AGING

- The shortening of telomeres correlates with aging
- The shortening of telomeres correlates with aging... and organisms may live longer if their telomeres are longer and their cells are capable of more cell divisions. Genetically engineered to over-express telomere binding protein

Length declines with age
Relationship between mean telomere length (± SEM, measured by T/S ratio using qPCR) and age at measurement in 99 zebra finches. Length declines with age Relationship between mean telomere length (± SEM, measured by T/S ratio using qPCR) and age at measurement in 99 zebra finches. The first sample (shown as year = 0) was collected at 25 d. The data are plotted at the individual level in Fig. S1. Heidinger B J et al. PNAS 2012;109: ©2012 by National Academy of Sciences

25 d and lifespan in zebra finches (n = 99).
Individuals with longer telomeres live longer Relationship between natural log-transformed relative telomere length (T/S ratio from qPCR) at 25 d and lifespan in zebra finches (n = 99). Relationship between natural log-transformed relative telomere length (T/S ratio from qPCR) at 25 d and lifespan in zebra finches (n = 99). Heidinger B J et al. PNAS 2012;109: ©2012 by National Academy of Sciences

And, short life correlates with shorter telomeres
Relationship between mean (± SEM) telomere length (T/S ratio from qPCR) and age at measurement (first sample, shown as year = 0, was collected at 25 d) in zebra finches in three lifespan categories. Green = mean lifespan of 1.6 yrs Red = mean lifespan of 3.6 yrs Black = mean lifespan of 6.3 yrs Relationship between mean (± SEM) telomere length (T/S ratio from qPCR) and age at measurement (first sample, shown as year = 0, was collected at 25 d) in zebra finches in three lifespan categories. Lifespan categories were created by dividing the 99 zebra finches into three equal groups on the basis of their age at death. Birds in the shortest lifespan group (n = 33) lived for an average of 1.6 y and are represented by the solid, green line; birds in the middle lifespan group (n = 33) lived for an average of 3.6 y and are represented by the dashed, red line; and birds in the longest lifespan group (n = 33) lived for an average of 6.3 y and are represented by the dotted, black line. Birds in the shortest and middle lifespan groups did not differ in telomere length at the first two sampling points (at 25 d, P = 0.99 and at 1 y, P = 0.85). Birds in the longest-lived group had consistently longer telomeres than those in the other two groups where sample sizes permitted comparisons (at 25 d, P < 0.001; at 1 y, P = 0.02; and at 3 y, P = 0.05). P values are based on Fisher's least significant difference (LSD) tests and significant differences, *P < 0.05. Heidinger B J et al. PNAS 2012;109: ©2012 by National Academy of Sciences

HOW might telomere length influence division of cells?

Telomere shortening drive p53 activation
Telomere shortening activates p53 and drives formation of epithelial cancers through gene amplification and deletion. Telomere shortening activates p53 and drives formation of epithelial cancers through gene amplification and deletion. Telomeres shorten progressively with cell division due to the end-replication problem in settings of insufficient telomerase, including in human fibroblasts, aging tissues, early cancers and diseases of high cellular turnover. Critical telomere shortening compromises the telomere cap and results in a DNA damage response that activates the p53 tumor suppressor protein. This activation of p53 induces replicative senescence in cultured human fibroblasts, impairs stem cell self-renewal, induces apoptosis in tissue progenitor cells, causes premature aging and strongly suppresses tumor formation. If p53 is mutated or deleted, these responses to telomere dysfunction are mitigated and chromosomal fusions are tolerated. The generation of fused chromosomes results in dicentric chromosomes (chromosomes with two centromeres) and when these attach to opposite spindle poles, chromosome breakage occurs. These broken ends serve as potent catalysts for translocations, focal amplifications and focal deletions. Such CNAs drive development of carcinomas and explain the widespread gene copy number changes seen in human cancers. Artandi S E , and DePinho R A Carcinogenesis 2009;31:9-18 © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

Trade-off between CANCER (high division) and AGING/TISSUE DAMAGE (no division)
Telomeres: - p53 is a tumor suppressor. - Tyner (et al. 2002): - mice who were deficient in p53 were susceptible to cancer (no repair). - They also isolated a strain that had overproduction of p53. These mutants had a reduce susceptibility to cancer, but they aged more rapidly than normal mice.

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