Objectives

1. Objectives • Essential Question: Why & how do our cells reproduce DNA? • Restate what replications is and why it’s important. • Describe the steps of DNA replication. • Compare the roles of DNA helicase, DNA polymerase, and ligase. • Compare the process of DNA replication in prokaryotes and in eukaryotes. • Illustrate the steps of replication in a drawing.

2. Vocabulary • DNA replication • DNA helicase • DNA polymerase

3. In the first section of this unit you learned what DNA is. The second section you learned what happens in G1: the process of gene expression; transcription and translation. You should also know that DNA is a large molecule built of billions of subunits. DNA Synthesis • These subunits are comprised of a phosphate group, a ribose sugar, and one of 4 nitrogenous bases. You should also recall that in DNA A bonds with T and G bonds with C.

4. Cells don’t last forever • Every cell in an organism has a particular life span… they don’t last forever. • Cells are born, grow and do their jobs, divide, and finally create two new daughter cells. • The life span is cyclical & with every new generation the cells that are formed carry out the functions of the parent cell because they are given each an exact copy of the parent cell’s DNA. • The cyclical life span of a cell is called the CELL CYCLE.

5. The Cell’s life. The cell Cycle

6. DNA Synthesis = DNA DUPLICATION • Every time a cell goes through a cycle it must DUPLICATE ITS DNA SO THAT WHEN IT MAKES NEW BABY CELLS THEY BOTH HAVE THE EXACT SAME DNA. • Happens in ‘S’ phase • In this section we are going explore the process of DNA synthesis, called DNA replication. • This process is extremely precise and an incredibly important.

7. From single-copy of each chromosome The Cell Cycle: The part we’re isolating. • To double-copy

8. What’s different about these cells? 1st Gap Phase DNA Synthesis Phase REPLICATION • These are identical strands of DNA • There are two copies formed in replication (S phase) because each are destined for the 2 identical new cells that are formed, called daughter cells, after the cell divides. • Identical DNA must be synthesized in order to achieve this. • The process of DNA synthesis is REPLICATION.

9. HOW IS DNA REPLICATED? • There are three ways that DNA can be duplicated… • Conservative: original is left completely original. • Semi-conservative: new DNA is half original and half new. • Dispersive: DNA is randomly duplicated. • The Meselson-Stahl experiment: • Showed that DNA is replicated semi-conservatively. • This means, the end product is one strand of original DNA and one strand of newly-formed DNA.

10. SO. What is the point of replication? The point of replication is to produce exact copies of the original cell’s DNA.

11. DNA Replication: THE STEPS • The process of replicating DNA is broken down into three major steps. • Your job is to know these 3 steps and be able to summarize what happens in each. • The three steps are: • Unwinding and Separating DNA Strands • Adding complimentary bases • Leading Strand: • Lagging Strand: RNA Primers attach • Formation of Two Identical DNA molecules

12. DNA Replication • Step 1: • DNA helicases unwinds & separates the original DNA double helix. • These proteins wedge themselves between the two strands of the double helix and break the hydrogen bonds between the base pairs. • Forms Replication Forks • As the double helix unwinds, the two complementary strands of DNA separate from each other and form a Y shape.

13. DNA Replication Step 2: New DNA is formed from DNA template. RNA Primers attach to specific regions. New nucleotides are added to the primer by the enzyme DNA Polymeraseaccording to the base-pairing rules. • DNA polymerases create the formation of the new complimentary DNA molecule by moving along each strand in a 5’ to 3’ direction. • They add nucleotides to a new daughter compliment in a 5’ to 3’ direction only! Primer Primer

14. DNA Replication 5’ You know that DNA is anti-parallel because of the bi-directionality of DNA. One side goes 3’ to 5’. The other 5’ to 3’. New nucleotides can only be added to the 3’ end of the existing chain. One side (the top pictured here) is the LEADING STRAND. It has its new strand continuously synthesized as helicase unwinds more DNA. The other side, the LAGGING STRAND (on bottom) is discontinuous replication because it the bases are oriented in the wrong direction. 5’ DNA polymerase can only add nucleotides in a 5’ to 3’ direction.

15. DNA Replication • Step 2: Leading strand • Primers attach. • Primers are pre-made sequences of RNA. These bind to complementary regions of the original DNA once it’s separated. • Primers serve as tethers from which replication can proceed from the 3’ end. • DNA Polymerase attaches free nucleotides to the 3’ end of the primer. • On the leading strand there is one primer and the newly forming DNA follows the replication fork as the new DNA is synthesized. Primer

17. DNA Replication • Step 2: Lagging strand • The lagging strand creates a particular problem for replication. • Since DNA can only be synthesized from the 3’ end of the primers, the lagging strand would be left incomplete. • Multiple primers attach to the lagging strand as the replication fork moves forward, creating what are known as “Okasaki Fragments” • These are usually 1-2000 nucleotides long.

18. DNA Replication • Step 2: Lagging strand • If you notice, the Okazaki Fragments are not joined together. • A special enzyme, call ligase, joins Okasaki fragments together to form one continuous molecule. Ligase Ligase

19. DNA Replication, continued Step 3: • The process completes when all the original bases have been paired with a new complementary nucleotide. • Each double-stranded DNA helix is made of one new strand of DNA and one original strand of DNA.

20. DNA Replication Click to animate the image.

21. Visual Concept: DNA Replication

22. Summary • What is DNA replication? • Why does it happen? • Know the steps… • On your handout, label: Where is… a. Leading Strand b. Lagging Strand c. Okazaki fragments d. DNA Ligase e. Replication Fork f. DNA Polymerase g. All 3’ & 5’ ends. h. Where helicase should be

23. Replication Proteins…Quality Control • Replication involves many proteins that form a machine-like complex of moving parts. • These proteins play a key role in making sure that the process is flawless. • DNA Helicase unwinds DNA carefully so it doesn’t get torn. • Ligase is a protein that ensures proper bonding of growing Okasaki fragments. • DNA polymerase adds complimentary nucleotides. • DNA polymerase also has a “proofreading” function. • During DNA replication, errors sometime occur and the wrong nucleotide is added to the new strand. • This could lead to cancer if not detected. • If a mismatch occurs, the DNA polymerase has the amazing ability of being able to backtrack, remove the incorrect nucleotide, and replace it with the correct one. • This decreases the chances of the wrong DNA being made, lessening our chances of CANCER!

24. Replication v Transcription • Based upon what you’ve learned about transcription and replication, shoulder partner share and come up with a similarity between transcription and replication. • WHAT IS THE SIMILARITY BETWEEN TRANSCRIPTION & REPLICATION? • Similarities: transcription and replication both use DNA as the template for copying. • SHOULDER PARTNER AND ANSWER: WHAT IS A CRITICAL DIFFERENCE BETWEEN REPLICATION AND TRANSCRIPTION? • Differences: • Intranscription, a new molecule of mRNA is made from the template DNA by using the enzyme RNA polymerase. • In DNA replication, a new molecule of DNA is made from the template DNA by using the enzyme DNA polymerase.

25. Objectives Primary • Compare the process of DNA replication in prokaryotes and in eukaryotes. • Identify the features of prokaryotic and Eukaryotic replication on an illustration. Secondary • Compare the number of nucleotides replicated in eukaryotes and prokaryotes by calculating the rates of replication.

26. Prokaryotic and Eukaryotic Replication • All cells have chromosomes, but eukaryotes and prokaryotes replicate their chromosomes differently. • The main difference between prokaryote and eukaryote replication is how many start sites each have. • Eukaryotes have hundreds of start sites. • The start sites then regulate how replication proceeds in each organism type.

27. Prokaryotic DNA Replication • Recall the structure of prokaryotic DNA. • Does anyone remember what the structure is? • Prokaryotic cells usually have a single chromosome which is a closed loop attached to the inner cell membrane. • Replication in prokaryotes begins at a single site along the loop.This site is called the origin of replication.

28. Prokaryotic Replication • Two replication forks begin at the origin of replication. • Replication occurs in opposite directions until the forks meet on the opposite side of the loop. • The result is two identical loops of DNA.

29. Eukaryotic Replication • Eukaryotic cells often have several chromosomes which are linear and contain both DNA and protein. • How many chromosomes do humans have? • What are the proteins called that hold DNA in nucleosomes? • Eukaryotic replication starts at many sites along the chromosome. • This process allows eukaryotic cells to replicate their DNA faster than prokaryotes.

30. Eukaryotic Replication • Two distinct replication forks form at each start site and replication occurs in opposite directions. • This process forms replication “bubbles” along the DNA molecule. • Replication bubbles continue to get larger as more of the DNA is copied. • The replication bubbles keep growing until they join together with other bubbles and complete replication. • Replication is complete when two identical complementary strands of DNA is formed.

31. Eukaryotic Replication Original DNA Replication bubbles Replication bubbles joining Original + newDNA New + originalDNA

32. Prokaryotic and Eukaryotic Replication • Even the smallest eukaryotic chromosomes are often 10 times the size of a prokaryotic chromosome. Eukaryotic chromosomes are so long that it would take 33 days to replicate a typical human chromosome if there were only one origin of replication. • As such, evolution has allowed Human chromosomes to replicate in about 100 sections that are 100,000 nucleotides long, each section with its own starting point. • Remember, the 46 human chromosomes laid end to end would measure ≈ 2m. Bacterial chromosomes measure ≈ 0.25cm! • Because eukaryotic cells have multiple replication forks working at the same time, an entire human chromosome can be replicated much faster, in only about 8 hours. • Bacteria replicate their small genomes in minutes…

33. In-class Activity: Replication Comparison • Label the letters in your notes. • Save this paper for a follow-up activity that will be turned in at the end of class. • Word-bank: Original DNA (x2), New DNA (x2), Replication Forks (x2), Replication bubble • What is the difference between prokaryotic replication & eukaryotic replication? • Why are they different? A C E B G F D

34. Summary • In DNA replication, the DNA molecule unwinds, and the two sides split. Then, new bases are added to each side until two identical sequences result. • The replication of DNA involves many proteins that form a machinelike complex of moving parts. • In prokaryotic cells, replication starts at a single site. In eukaryotic cells, replication starts at many sites along the chromosome.

35. Replication concept Check • 1. What is the purpose, outcome of DNA replication AND what stage does it happen in the cell cycle? • To create an identical, duplicate copy of original DNA, “S” • 2. What enzyme is responsible for “unzipping” the DNA double helix? • DNA Helicase • 3. What enzyme is responsible for adding nucleotides to the “unzipped” DNA? • DNA polymerase • 4. What is the enzyme responsible for bonding the fragments on the lagging strand? • Ligase • 5. (Review) What is the enzyme responsible for creating mRNA copies of genes in DNA? • RNA polymerase • 6. What is the enzyme responsible for “proofreading” the newly made DNA, checking for mismatched base-pairs? • DNA polymerase

36. Summary… The More Complex Version