1. Bio 402/502 Section II, Lecture 2 Nuclear Processes: DNA replication and transcription Dr. Michael C. Yu

2. Purpose of DNA replication? General concepts of DNA replication How does a cell accomplish this? Biological significance of DNA replication? Cells die if they can’t replicate their DNA properly Duplicate a cell’s genetic material - with accuracy - why? Via a complex set of cellular machineries - how do you identify these proteins? Rate: as high as 1KB per second - difference eukaryotic cells have different genome size

3. Prokaryotic vs. eukaryotic DNA replication Prokaryotes: Single origin of DNA replication (circular chromosome) Eukaryotes: Multiple origins of DNA replication Occurs during S phase of the cell cycle

4. DNA replication is “semi-conservative” (Figure obtained at www.sparknotes.com) DNA replication: 1)Separation of the two strands 2)Complete replication using each strand as a template for the synthesis of a new “daughter” strand

5. DNA replication is also bi-directional Replication starts at “origins of replication” or “replication fork” Can have multiple origins within a chromosome - efficient

6. Identify origins of replication: using yeast as a model organism (Figure obtained from Alberts, 4th ed.)

7. DNA polymerase: the enzyme that makes DNA Many more DNA polymerases Eukaryotes: Five DNA polymerases: I, II, III, IV, and V Can be broadly categorized into replication or repair Prokaryotes (E. coli): Substrate: deoxyribonuceloside triphosphate

8. Common structure of DNA polymerase Enzyme has independent domains Conserved sequence motif for catalytic active site Responsible for positioning template correctly at the active site Binds DNA as it exits the enzyme

9. Understanding the function of domains within DNA polymerase In vivo mutagenesis? In vitro assays? Create mutations within the domain and test its function in vivo Biochemical reconstitution of replication assay (bacteria) Not really able to do so with eukaryotes - too complex.

10. Process of DNA replicatiton 1. Helicase separates both strands of the DNA 2. Single-stranded proteins bind and maintain separated strands 3. Prime with 3’-OH end (difference between leading & lagging strand) 4. Synthesis of DNA by DNA polymerase 5. Ligation of Okazaki fragments by ligase (lagging strand only) (Figure obtained at Ohio State Biosci website)

11. A different view of DNA replication process Things to also consider: chromatin access by trans-factors

12. How do you identify “essential” genes involved in DNA replication? The use of conditional mutants - (e.g. temperature sensitive) Good model organisms: yeast and bacteria 30°C 37°C Mutagenized cells grown on petri dish Loss of CFU at non-permissive temperature In vitro DNA synthesis

13. Transcription by RNA polymerase II: making of messenger RNAs Only one of the two strands of DNA is transcribed into mRNA 3’ 5’ 5’ 3’

14. Transcription in Prokaryotes Lacks nucleus: couples transcription with translation

15. Transcription in the eukaryotes Major players involved in regulating transcription are transcription factors The predominant form of eukaryotic gene expression Several types - basal, activators, co-activators, etc. Three RNA polymerases in eukaryotes: RNA Pol I: 5.8S, 18S, and 28S rRNA genes RNA Pol II: all protein-encoding genes, snoRNA genes, some snRNA genes RNA Pol III: tRNA genes, 5S rRNA genes, some snRNA and small RNA genes Cell/tissue specific - achieve cell/tissue specificity

16. Basic Concept of Eukaryotic Gene Expression Activation of gene structure (i.e. chromatin) Transcription initiation Process of transcripts Export of mRNAs to the cytoplasm Translation of mRNAs to proteins Majority of the eukaryotic gene expression controls Transcription elongation

17. Basic Concept of Eukaryotic Gene Expression What is the role of chromatin during transcription? Euchromatin vs. heterochromatin First step - open up chromatin structure

18. General structure of chromatin resulting in its compactness

19. Subunit of all chromatin: nucelosome (Lewin, Genes IX) A nucleosome wraps approx. 200bp DNA How do you know if a segement of DNA is protected/wrapped within a nucelosome? Difference in their susceptibility to MN A nucleosome is composed of DNA & histones

20. Histone N-terminal tails are post-translationally modified Two copies of each core histones per nucleosome N-terminal tails of histones are positioned outside of a nucleosome (Lewin, Genes IX)

21. Model of chromatin regulation during transcription Histone PTMs: a major control in chromatin structure Modification status indicative of transcriptional state Which histone modifications correlates with gene activation or silencing? (Li et al, Cell, 2007)

22. Initiating transcription at the promoter region is a multi-step process (Lewin, Genes IX) Histone modifications and their implications on gene expression: Acetylation- transcriptionally active Methylation (K,R)- transcriptionally silent Phosphorylation (S) - activation Ubiquintination - signals methylation Sumolyation - transcriptionally silent Normally, chromatin is silent

23. Events leading to transcription initiation at the promoter (Lewin, Genes IX) 1. Recruitment of activator (trans) to the cis-element 2. Activators recruit chromatin remodelers 3. Modification of chromatin, reorganization of nucleosome, release of chromatin compactness

24. Recognition of core promoter elements by transcription factors (Thomas & Chiang, 2007) Selectivity of promoters determines which transcription factors are recruited to activate gene transcription. How would you determine the consensus promoter sequences of a gene?

25. Identify cis-acting elements using mutagenesis approach Approaches: linker scan, alanine substitution, deletion, etc. (Hou et al, 2002) a) Linker scan approach b) Assays to determine the effect of mutation

26. Recruitment of RNA polymerase II: start of transcription initiation (Lodish et al, 2000) Transcription factors specifies the location of transcription and recruits RNA pol II. How would you determine which factors are responsible for transcription in a cell?

27. Protein-protein interactions between transcription factors Principles of a two-hybrid assay: Pros & cons: False positives More in vivo context Let cells do the work Organism specific (lacking PTM) Use a known transcription factor as a “bait”

28. Identifying protein-protein interactions between transcription factors Detect biochemical association between proteins using Co-IP Can also use epitope-tagged proteins such as Myc, HA, FLAG, etc Use mass spec to identify co-IP’ed proteins

29. Modes of transcription factor activation (Lewin, Genes IX) How would you determine binding of a TF to a promoter?

30. Detecting DNA-protein interactions in vitro Can use EMSA (electrophoresis mobility shift assay) or gel- shift assay: Use of EMSA involved biochemically purified components/extracts An example of EMSA Radio-labeled DNA probes (i.e. specific promoter elements) (Sigma website) or PAGE How would you use this method to determine which mode of transcriptional activation

31. 1. Crosslink Protein-DNA complexes in situ 2. Isolate nuclei and fragment DNA (sonication or digestion) 3. Immunoprecipitate with antibody against target nuclear protein and reverse crosslinks 4. Identify DNA sequence by quantitative or real-time PCR Detecting DNA-protein interactions in vivo 5. Detection of PCR products by PAGE or real-time machine