1. DNA REPLICATION Revising DNA structure Why replicate? Semiconservative replication Replication - overview Leading strand Lagging strand

2. Central Dogma of Information Flow

3. What is DNA replication? DNA replication is the synthesis of DNA using itself as a template. Three stages: 1. Initiation 2. Elongation 3. Termination

4. DNA Nucleotide O O=P-O O Phosphate Group Group CH2 O C C CC Sugar Sugar(deoxyribose) N Nitrogenous base (A, G, C, or T) (A, G, C, or T)

5. DNA Double Helix P P P O O O 1 2 3 4 5 5 3 3 5 P P P O O O 1 2 3 4 5 5 3 5 3 G C TA

6. When does DNA replication take place? Every time cells need to duplicate themselves: During growth During growth During embryo development. During embryo development. To repair damaged tissues, e.g. after an injury or wound. To repair damaged tissues, e.g. after an injury or wound. To replace damaged or dead cells. To replace damaged or dead cells. DNA replication takes places only once in a cell’s cycle, BEFORE cell division. DNA replication takes places only once in a cell’s cycle, BEFORE cell division.

7. Complementary base pairing allows each strand of DNA to serve as a template for DNA replication. After replication is completed, two identical DNA molecules are obtained. DNA is a perfect illustration of function following form (structure dictates function).

8. Something old and something new During DNA replication, both strands are “copied”, that is, each one acts as a model to make a new strand. SEMICONSERVATIVE This type of replication is known as SEMICONSERVATIVE, since in each DNA molecule there is always an old strand, the original template and a new strand recently made.

9. DNA replication is semiconservative

10. Replication I Replication can be summarised in 3 steps: 1) Separate the original strands 2) Use each strand as a template and make a new complementary strand using free nucleotides. 3) Join the nucleotides in each strand to form a chain. As a result, 2 identical DNA molecules are obtained.

11. Replication II: the players DNA replication is a very complex process involving several proteins and enzymes. In bacteria, these are: The helicase The helicase The topoisomerases The topoisomerases The SSBP The SSBP The polymerases I y III The polymerases I y III The primase The primase The ligase The ligase

12. How does it happen? 1. STRANDS ARE SEPARATED. The helicase unwinds the double helix, separating both strands by breaking the H bonds between complementary bases. Next, the SSBPs hold each strand in place. Why?

13. The DNA polymerase III is the enzyme that, according to the sequence in the template brings the complementary nucleotides adding them to form a new strand. Example: Sequence in the template strand:A-T-T-C-C-G-A-T- … Complementary new strand formed: T-A-A-G-G-C-T-A- … REMEMBER COMPLEMENTARY BASE PAIRING: A-T & C-G. This happens in both template strands so that two “models” are being copied at the same time. However, …. 2. SYNTHESIS OF NEW STRANDS

14. HOWEVER, there is a tiny detail to take into account… REMEMBER THE DNA STRUCTURE. Strands are named according to the last free group bound to the pentose in each end: either a phosphate in C5’ or a OH in C3’. Therefore strands are named as 5’ 3’ and its complementary: 3’ 5’. DNA strands are said to be ANTIPARALLEL.

15. How to copy in opposite directions: THE LEADING STRAND AND THE LAGGING STRAND Solution: first one strand is copied in one sense and next the other is copied in the opposite one. The strand that gets copied first is known as the leading strand and the second that gets copied next is known as the lagging strand. So what is the difference between the leading strand and the lagging strand? Simple: which one is copied first. Problem Problem: olymerase III can only add nucleotides in the direction 5’ – 3’ Polymerase III can only add nucleotides in the direction 5’ – 3’. This means that the new strand will be formed in the direction 5’ – 3’. So, where to start?

16. 3. HOW IS EACH STRAND COPIED ? Since the DNA polymerase III acts in the sense 5’—3’, the strand that has a free OH (in the sense 3’—5’) will be acting as the leading strand.* The leading strand is copied continuously. The other strand (5’- 3’ sense) is copied next. Since it is in the opposite direction, the polymerase will go slower and in chunks. The lagging strand is copied discontinuously in segments called Okazaki fragments *Pay attention to the templates (in blue): where replication starts and in what direction.

17. 4. What is the DNA polymerase III for? DNA polymerase III always acts in the 5’—3’ sense but it needs a small segment of double strand to start adding nucleotides. This “starter fragment” known as primer consists of RNA and is made by an enzyme called primase. Once the primer is in position, polymerase III just makes a new strand adding complementary nucleotides. This takes place both in the leading and lagging strands.

18. So far … In the leading strand, in the 3’—5’ sense, the DNA polymerase III adds complementary nucleotides continuously to make a new strand starting with a RNA primer. In the leading strand, in the 3’—5’ sense, the DNA polymerase III adds complementary nucleotides continuously to make a new strand starting with a RNA primer. On the other hand, in the lagging strand (in the 5’—3’ sense) replication is discontinuous. The DNA polymerase III adds complementary nucleotides as well, starting with a RNA primer, but in chunks called the Okazaki fragments. On the other hand, in the lagging strand (in the 5’—3’ sense) replication is discontinuous. The DNA polymerase III adds complementary nucleotides as well, starting with a RNA primer, but in chunks called the Okazaki fragments.

19. 5. On we go … Once the Okazaki fragments are synthesised on the one hand and the new continuous strand on the other, there’s the final proof-reading to check if any mistakes were made and the removal of the RNA primers, which are no longer necessary. This is done by the enzyme DNA polymerase I, that: Removes the RNA primers. Removes the RNA primers. Adds a few more nucleotides to the Okazaki fragments, elongating them. Adds a few more nucleotides to the Okazaki fragments, elongating them. Checks that newly synthesised strands are error- free (correct base pairing and no mismatches). Checks that newly synthesised strands are error- free (correct base pairing and no mismatches).

20. 6. Finally… The only thing left is to join all the Okazaki fragments and the free nucleotides in the continuous strand. This is done by the ligase, which forms phosphodiester bonds between the P- group of the first fragment and the OH from the following fragment.

21. Understood? Check the ends in both template strands WARNING! To simplify diagrams, in books 2 DNA pol III are usually drawn making the new strands, but in fact it is only one.

23. Some animations http://www.youtube.com/watch?v=xnT_OuAYfvY DNA song http://www.youtube.com/watch?v=teV62zrm2P0&feature=related DNA replication 1 http://www.youtube.com/watch?v=hfZ8o9D1tus DNA replication 2