1. DNA Replication Lecture 7

2. DNA Replication  Synthesis of two new DNA duplexes based on complementary base sequences with parental DNA.  Is progressive, involves alternation in unwinding and rewinding of DNA duplex.  Rate of synthesis (bp/sec) depends on the complexity of organism.  Controlled at several check points.  Mediated by many enzymes, and other cellular factors.

3. 1. Semi-Conservative Replication  One-half of each new molecule of DNA is old (template strand)  One-half of new molecule of DNA is new (complementary strand)

4.  DNA replication in:  Eukaryotes: may be initiated at more than one site, each with its own replication origin (bidirectional replication)  Prokaryotes and bacteriophage: one replication origin for initiating DNA synthesis.  The two anti-parallel DNA strands are replicated in different ways: DNA polymerases extend DNA strands only in the 5'  3' direction:  Leading strand: continuously synthesized in 5'  3' direction.  Lagging Strand: synthesized in 5'  3' direction, but discontinuously as Okazaki fragments, which are joined together by DNA ligase. 2. DNA Replication

5. 3. DNA Strand Replication

6. 4. Enzymes of Replication  DNA gyrase (topoisomerase II)  Helicases: separation of DNA strands  Single-stranded binding (SSB) proteins: preventing newly- exposed single-stranded DNA from re-an­nealing.  Primase: synthesis of RNA primers  DNA polymerases: synthesis of new DNA and DNA proof-reading  DNA Polymerase I: filling in gaps between Okazaki fragments formed during lagging-strand synthesis  DNA Polymerase II: No specific task assigned in DNA replication  DNA Polymerase III: the main polymerizing agent  DNA ligase: Covalently link successive Okazaki fragments.

7. 5. Helicases  The two DNA strands do not spontaneously come apart during DNA replication, but need to be separated by helicases,  Helicases bind ATP and single- stranded sections of DNA, and move progressively forward.  Helicases unwind DNA in advance of DNA polymerase binding.

8. 6. Single-Stranded DNA Binding Proteins  The new single-stranded DNA regions are quickly covered by specific single- stranded DNA binding proteins (SSB)  SSB prevent coiling of single-stranded DNA, and make bases available for reading and matching with complementary base.  SSB do not bind ATP, and have no known enzymatic function.  The binding of one SSB promotes the binding of another to the next section of a single-stranded DNA (cooperative binding).

9. 7. Sealing Single-Strand Cuts by DNA Ligase  DNA ligase catalyze joining of Okazaki fragments in an energy- dependent process.  The free energy required by this reaction is obtained through the coupled hydrolysis of:  NAD + → NMN + + AMP  ATP → PPi + AMP.

10. 8. Initiation of Okazaki Fragments  The primers for Okazaki fragments are made by primases, which recognize specific DNA sequences along single-stranded DNA.  Primase forms a complex with 6-7 other polypeptides to form primosome.  The primosome moves 5' → 3' on the lagging-strand to prime the initiation of Okazaki fragments.  Movement of the primosome along DNA leads to SSB displacement, the recognition of start sites, and the polymerization of nucleotides into RNA.  Removal of RNA primer occurs by DNA polymerase I, which also fills the gaps between RNA-primed DNA fragments.  RNA primers prevent mistakes near the starting points of DNA chains.

11. 9. DNA Polymerase  There are different forms of DNA polymerase  DNA polymerase III is responsible for the synthesis of new DNA strands  DNA polymerase is actually an aggregate of several different protein subunits, so it is often called a holoenzyme  Primary job is adding nucleotides to the growing chain  Also has proofreading activities

12. 10. DNA Polymerase III  The active form of DNA polymerase III is an assembly (holoenzyme) of seven polypeptides, needed for correct biological activity:  One of the polypeptides carries 5' → 3' exonuclease and polymerizing functions  Another polypeptide has the 3' → 5' exonuclease activities.  Additional polypeptides bind ATP, needed for DNA polymerase III to start synthesis at the end of RNA primer.  Once DNA polymerase III begins polymerization, it continues until the end of its template is reached.  Two copies of the DNA polymerase III holoenzyme are present at the replicating fork, so that the complex can carry out the synthesis of both the leading and lagging strands.

13. 11. Beginning: Origins of Replication  Replication begins at specific sites called origins of replication  In prokaryotes, the bacterial chromosome has a specific origin  In eukaryotes, replication begins at many sites on the DNA molecule  100’s of origins  Proteins that begin replication recognize the origin sequence  These enzymes attach to DNA, separating the strands, and opening a replication bubble  The end of the replication bubble is the “Y” shaped replication fork, where new strands of DNA elongate

14. 11. Beginning: Origins of Replication

15. 12. Elongation  Elongation of the new DNA strands is catalyzed by DNA polymerase  Nucleotides align with complimentary bases on the template strand, and are added by the polymerase, one by one, to the growing chain  DNA polymerase proceeds along a single-stranded DNA molecule, recruiting free nucleotides to H-bond with the complementary nucleotides on the single strand  Forms a covalent phosphodiester bond with the previous nucleotide of the same strand  The energy stored in the triphosphate is used to covalently bind each new nucleotide to the growing second strand  Replication proceeds in both directions

16. 12. Elongation

17. Ligase  Catalyzes the formation of a phosphodiester bond given an unattached adjacent 3‘ OH and 5‘ phosphate.  This can join Okazaki fragments  This can also fill in the unattached gap left when an RNA primer is removed  DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.

19. Primers  DNA polymerase cannot start synthesizing on a bare single strand.  It only adds to an existing chain  It needs a primer with a 3'OH group on which to attach a nucleotide.  The start of a new chain is not DNA, but a short RNA primer  Only one primer is needed for the leading strand  One primer for each Okazaki fragment on the lagging strand

20. Primase  Part of an aggregate of proteins called the primeosome.  Attaches the small RNA primer to the single-stranded DNA which acts as a substitute 3'OH so DNA polymerase can begin synthesis  This RNA primer is eventually removed by RNase H  the gap is filled in by DNA polymerase I.

22. Ending the Strand  DNA polymerase only adds to the 3’ end  There is no way to complete the 5’ end of the new strand  A small gap would be left at the 5’ end of each new strand  Repeated replication would then make the strand shorter and shorter, eventually losing genes  Not a problem in prokaryotes, because the DNA is circular  There are no “ends”

23. Telomeres  Eukaryotes have a special sequence of repeated nucleotides at the end, called telomeres  Multiple repetitions of a short nucleotide sequence  Can be repeated hundreds, or thousands of times  In humans, TTAGGG  Do not contain genes  Protects genes from erosion thru repeated replication  Prevents unfinished ends from activating DNA monitoring & repair mechanisms

24. Telomerase  Catalyzes lengthening of telomeres  Includes a short RNA template with the enzyme  Present in immortal cell lines and in the cells that give rise to gametes  Not found in most somatic cells  May account for finite life span of tissues

26. Proteins of DNA Replication

27. Summary View of Replication

28. 1. Why is replication necessary? 2. When does replication occur? 3. Describe how replication works. 4. Use the complementary rule to create the complementary strand: A--- ? G--- ? C--- ? T--- ? A--- ? G--- ? A--- ? G--- ? C--- ? A--- ? G--- ? T--- ? Replication Quiz

29. 1. Why is replication necessary? So both new cells will have the correct DNA 2. When does replication occur? During interphase (S phase). 3. Describe how replication works. Enzymes unzip DNA and complementary nucleotides join each original strand. 4. Use the complementary rule to create the complementary strand: A--- T G--- C C--- G T--- A A--- T G--- C A--- T G--- C C--- G A--- T G--- C T--- A Replication Quiz