DNA Duplication and Sequencing

1. DNA Duplication and Sequencing Genetics Spring 2014

2. Outline

3. DNA Replication – Different Forms of DNA Circular DNA found in bacterial chromosome or as plasmid (in bacteria and eukaryotic cells) exist as single replicon with one origin of replication. Linear DNA found in eukaryotic chromosomes can contain up to thousand origins of replication. Slightly different modes of replication

4. DNA Replication – semiconservative mode in E. coli Predictions of semi-conservative replication in the Meselson-Stahl experiment using 14N/15N and CsCl equilibrium density gradient centrifugation (1958). See fig 6.3 for original data DNA form E.coli, circular DNA

5. DNA Replication – semiconservative mode in eukaryotes Use BrdU (Bromodeoxyuridine), analogous to thymidine and that can be detected under light microscopy. The two BrdU DNA are very fluorescent , quench each other and thus appear dark (X-rays induced more breakage and sister-chromatid exchange than usual).

6. DNA Replication – Origins of replication q (theta) replication on circular DNA • Replication can be uni- or bi-directional . • During q-form replication, both parental DNA strands remain intact. E. coli chromosome

7. Rolling-circle replication on circular DNA During rolling circle replication, one of the template DNA strands is cut to create a primer 3'-OH end. Phage lambda; F factor of E. coli

8. One strand of DNA is nicked and the free 3’-OH is extended by the DNA polymerase. The 3’ end on the circle is lengthened while the growing point rolls around the circular template. The 5’ end is displaced and forms a tail of single-stranded DNA that extends from the circle. Single-stranded tail is converted into double-stranded DNA by synthesis involving RNA primers as in the synthesis of the lagging strand of normal DNA synthesis. Then circular shapes are formed.

9. Continued rolling-circle replication results in a linear extension that contains tandem repeats of sequences found in the parental circular molecule Each complete replication around the circle adds another tandem repeat to the tail.

10. Multiple Origins of Replication on linear DNA Each replication bubble is bidirectional and as the bubbles grow larger, the replication forks of adjacent bubbles fuse where they meet.

11. DNA Replication – Various steps Unwinding, Stabilization and Stress relief Gyrase (topoisomerase), SSB proteins and helicase are key proteins in forming the replication fork.

12. The DNA gyrase introduces a double-stranded break ahead of the replication fork and swivels the cleaved ends around the central axis to relieve the stress of helix unwinding.

13. Initiation by the Primosome Complex • The primers are synthesized by Primasein prokaryotes and Primosomein eukaryotes. • Primosomecontains DNA polymerase alpha that first synthesizes primers (~ 12 NTPs) and then switches to synthesizing DNA (~ 35 dNTPs). • After that, the main DNA polymerase delta (eukaryotes) or DNA polymerase III (prokaryotes) takes over.

14. Chain Elongation and Proofreading - DNA synthesis always proceeds in a 5' to 3' direction. - Addition of a deoxynucleotide to 3'-OH end of a primer chain by the main DNA polymerase. -The DNA polymerase uses its exonuclease activity (3’ to 5’) to remove any mismatched base pair.

15. Discontinuous Replication of the Lagging Strand • While the leadingstrand is synthesized continuously, the laggingstrand is synthesized discontinuously (always follows the 5’ to 3’ direction on the newly-synthesized DNA fragment). • The okazakifragments are the results of the discontinuous synthesis.

16. Joining of the Okazaki Fragments Removal of RNA primers by the DNA polymerase delta (eukaryotes) or DNA polymerase I (prokaryotes). Replacement of primers with DNA polymerase delta (eukaryotes) or DNA polymerase I (prokaryotes). Joining adjacent DNA fragments by the ligase.

17. DNA Sequencing – Terminator Sequence Dideoxynucleotides lack a 3'-OH and are chain terminators

18. Sequencing takes advantage of ddNTP chain terminators. Each ddNTP has different colored fluorescent dye. The sequence is read from the newly synthesized DNA. The sequence is the complement of the template DNA.

19. DNA sequencing use fluorescent dideoxynucleotides Electropherogram reads 5’ to 3’ on the coding strand.

20. Dideoxynucleotides are anti-viral compounds because they inhibit viral replication • Retroviruses (HIV): AZT, ddI, ddC • Herpes simplex: inhibitor Acyclovir Scanning electron micrograph of human immunodeficiency virus (HIV), grown in cultured lymphocytes. Section electron microscopy of infected cell culture with Herpes simplex virus

21. DNA Sequencing – Next Generation Improvements in the rate of DNA sequencing over the past 30 years and into the future. From slab gels to capillary sequencing and second-generation sequencing technologies, there has been a more than a million-fold improvement in the rate of sequence generation over this time scale.