DNA replication

SBI4U0- DNA Replication Cells need to have the ability to reproduce for many reasons. When cells make copies of themselves, they must ensure that their entire genetic code is present in both the old and new cell DNA must replicate to ensure 2 complete copies of the genetic code. This is why cells must undergo the process of MITOSIS (remember, sister chromatids are copies of the same chromosome!)

DNA Replication During cell division, the genetic material has to be divided up between daughter cells or gametes. (Mitosis - production of daughter cells; Meiosis- production of gametes) DNA replicates first in INTERPHASE

Remember This? The Cell Cycle

The Cell Cycle Two Main Stages Interphase Cell Division Growth 1, Synthesis, Growth 2 Cell Division Mitosis, Cytokinesis

THE CELL CYCLE G1 G2 S Telophase Anaphase Cytokinesis Metaphase Prophase G1 G2 S

INTERPHASE Period between cell divisions Cell undergoes growth, replication of DNA, obtaining energy, making hormones, repairing damage, fighting disease *Most of the cell’s time is spent in this phase – length varies depending on the organism and cell type

Prophase

Metaphase

Anaphase

Telophase

Back to Interphase S Phase How does DNA replicate?

THE CELL CYCLE G1 G2 S Telophase Anaphase Cytokinesis Metaphase Prophase G1 G2 S

DNA Replication Produces two identical copies of the chromosome during S phase of interphase Catalyzed by many enzymes

DNA Structure and composition: DNA strands are antiparallel, meaning they run in opposite directions One strand runs in the 5'  3' direction, the other in the 3'  5' direction

DNA Structure and composition: The 3' end of one strand ends with the –OH on C3 (3rd carbon of the sugar molecule) The 5' end of a strand ends with a phosphate group attached to the C5 (5th carbon of the pentose sugar)

DNA Replication Watson and Crick’s model of DNA structure suggested how DNA could replicate Hydrogen bonds break Helix unzip Each strand act as a template to build new strand At this time there were no experimental results to support this hypothesis

3 models of replication: Semiconservative replication would produce molecules with both old and new DNA, but each molecule would be composed of one old strand and one new one. Conservative replication would leave intact the original DNA molecule and generate a completely new molecule. Dispersive replication would produce two DNA molecules with sections of both old and new DNA interspersed along each strand.

Semiconservative Model DNA begins by unzipping itself  the hydrogen bonds break between strands 2 strands separate, and each acts as a template or guide for directing the synthesis of the new strand. "Semiconservative" is used to describe this process since each new DNA molecule contains 1 old strand and 1 new strand This model was proven by Meselson & Stahl

Meselson and Stahl Experiment Meselson and Stahl concluded that DNA replication is not conservative but semiconservative Each strand acts as template for the building of the complementary strand Each DNA strand is composed of one parent strand and one newly synthesized strand

Meselson-Stahl experiment E. coli bacteria was grown using 15N, a denser isotope of N, for several generations. When 15N DNA was centrifuged in cesium chloride, which forms a density gradient, all the DNA settled in one single layer, forming a band.

M and S then switched to 14N for one generation. If DNA replication was conservative, they should have seen two bands, one containing the old DNA with 15N, the other containing the new DNA with 14N. If DNA replication was semiconservative, they should have seen one intermediate band.

After two generations with 14N, they saw two bands: One intermediate band (mix of 15N and 14N) One lighter band (only 14N) Over several generations, they found that the intermediate band did not go away even after they stopped using 15N. This indicates that the original 15N DNA was still there, still acting as a template.

Meselson-Stahl experiment

Video Overview http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio22.swf::Meselson and Stahl Experiment

DNA Replication

Steps Separating the DNA strands Building Complementary Strands Linking of Nitrogenous Bases and Proof-reading

1) Separating the DNA strands The replication of DNA must take place in many steps. The entire DNA strand cannot be replicated at once because the unraveled DNA would be too large for the cell. The piece of unwould DNA that is being replicated creates a DNA Replication Bubble At each end of the bubble is the Replication Fork The enzyme helicase breaks the hydrogen bonds holding the two complementary parent strands together, resulting in an unwound, unzipped helix that terminates at the replication fork. The enzyme gyrase / topoisomerase relieves any tension from the unwinding of the double helix

1. Separating DNA Strands Initiated at sites along DNA called Replication Origins – made of specific nucleotide sequences Enzymes and other proteins work together to unwind and stabilize the double helix

The Players: DNA helicase DNA gyrase (aka topoisomerase) SSBs (single-strand binding proteins)

DNA Helicase Recognizes specific nucleotide sequence (origin of replication) Unwinds double helix by breaking H bonds between complementary base pairs Opens up one or more replication bubbles

DNA Gyrase / Topoisomerase Relieves the tension produced by the unwinding of DNA Cuts one or two of the strands near the replication fork so that they can untangle and rejoin Topoisomerase in action: http://www.youtube.com/watch?v=EYGrElVyHnU

Single-stranded Binding Proteins SSBs Keep separated DNA strands apart by blocking hydrogen bonding Keep the templates (single DNA strands) straight

DNA Replication – Step 3 Single-stranded binding proteins (SSB’s) anneal to the newly exposed template strands, preventing them from reannealing by blocking hydrogen bonding.

1. DNA unwinding and unzipping 1. The enzyme helicase breaks the H-bonds holding the two complementary parent strands together, resulting in an unzipped helix that terminates at the replication fork. 2. The enzyme gyrase relieves any tension from the unwinding of the double helix. 3. Single-stranded binding proteins (SSBs) anneal to the newly exposed template strands, preventing them from re-annealing.

DNA Replication - Overview

2) Elongation: Building of Complementary Strands Replication begins in two directions from the origin Toward direction of replication fork on one strand Away from direction of replication fork on the other DNA polymerase builds new strands DNA polymerase III used in prokaryotes DNA polymerase III can only synthesize DNA in the 5’ to 3’ direction

2. Building complementary strands Because there may be more than one origin of replication in eukaryotes, more than one replication fork may exist Link-replication animation: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio23.swf

The Players Primase DNA polymerase DNA ligase

Primase DNA nucleotides can only be added by DNA polymerase to an existing strand. Primase builds RNA primers which are used to initiate DNA replication

DNA Replication DNA polymerase III cannot initiate a new strand by itself so a RNA primer is required The enzyme primase lays down RNA primers that will be used by DNA polymerase III as a starting point to build the new complementary strands.

DNA polymerase III Takes free nucleotides found within the cell and adds them in the 5’ to 3’ direction, first to the RNA primer and then to the DNA nucleotide that was just added The parent strand is used as a template

Leading (good guy) strand The daughter strand that grows continuously towards the replication fork as the double helix unwinds Occurs quickly Requires only a single RNA primer at replication origin

Leading Strand DNA polymerase III adds the appropriate deoxyribonucleoside triphosphates to the 3 prime end of the new strand using the template strand as a guide. The energy in the phosphate bonds is used to drive the process. The leading strand is built continuously toward the replication fork.

Lagging (scumbag) strand The 3’ to 5’ parent strand is a problem for DNA polymerase since it must synthesize in the 5’ to 3’ direction!

Lagging strand Built in short segments (in the 5’ to 3’ direction) away from the replication fork Requires many RNA primers

Lagging Strand A lagging strand composed of short segments of DNA, known as Okazaki fragments, is built discontinuously away from the replication fork.

DNA polymerase I DNA ligase Removes the RNA primers once they have been used and replaces them with the appropriate DNA sequence DNA ligase Joins the Okazaki fragments into one strand by the creation of phosphodiester bonds

Replacing the RNA Primers DNA polymerase I excises the RNA primers and replaces them with the appropriate deoxyribonucleotides. DNA ligase joins the gaps in the Okazaki fragments by the creation of a phosphodiester bond.

Link-leading vs lagging animation: http://highered. mcgraw- hill Link-leading vs lagging animation: http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::53 5::/sites/dl/free/0072437316/120076/micro04.swf

3) Quality Control DNA polymerase III acts as a proof- reader by checking the newly synthesized strand for any incorrectly inserted bases If a mistake is found, it backs up and replaces the incorrect base with the correct one

DNA Replication – Final Step! Errors missed by DNA polymerase III is “proofread” and repaired by DNA polymerase II by excising incorrectly paired nucleotides at the end of the complementary strand and adding the correct nucleotides. Similar repair mechanisms also help to repair the damage caused by carcinogens (toxic chemicals, UV light and other radiation)

Termination The process of DNA replication ends when all the replication bubbles meet. The DNA molecules rewind to gain their normal helical form. There is a problem though, due to the removal of primers- a little bit of a gap always remains on the lagging strand Therefore, entire chromosome can never be fully copied! It is estimated that with each round of duplication, around 100 bps of DNA are lost To deal with this problem, each chromosome has a series of bases the essentially code for nothing tagged onto the end of the chromosomes. These buffer ‘ends’ are called Telomeres. Courtesy of the enzyme ‘telomerase’ Slow erosion of the telomeres (after approximately 50 replications) leads to the aging and death of the cell.

Animations DNA Replication Fork: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf How Nucleotides are Added: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio23.swf

DNA Replication Simulator: http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

Telomeres, Aging and Cancer It is interesting to note that older people have shorter telomeres than younger people at the end of their chromosomes. Certain types of cells have no apparent shortage to their telomeres ex. Sperm and cancer cells. Follow the links below to gather more information on telomeres: http://learn.genetics.utah.edu/content/begin/traits/telomeres/ http://longevity.about.com/od/researchandmedicine/p/telomeres.htm