1. 21-1 Chapt 21 DNA Replication II: Mechanisms; telomerase Student learning outcomes: Describe how replication initiates –proteins binding specific DNA sequences Explain general elongation: –coordination leading, lagging strands Describe basic features of termination Describe basic features of telomerase Important Figs: 1, 7, 11, 18*, 19, 25, 26, 29*, 30, 31*, 32, 34, 36 Review problems: 1-5, 8, 11, 14*, 16, 21, 22, 23, 24, 25*

2. 21-2 21.1 Initiation and Priming in E. coli Initiation of DNA replication requires primers: Different organisms use different mechanisms for primers Primosome - - proteins needed to make primers to replicate DNA (A. Kornberg) E. coli primosome: –DNA helicase (DnaB) –Primase (DnaG) Primosome assembly at origin of replication, oriC is multi-step

3. 21-3 DnaA binds to unique oriC at sites called dnaA boxes; cooperates with RNAP, HU protein to melt nearby DNA region DnaB binds to open complex, facilitates binding of primase to complete primosome; DnaB helicase activity unwinds DNA Primosome remains with replisome, repeatedly primes Okazaki fragment synthesis on lagging strand E. Coli primosome: Priming at oriC: primase

4. Key proteins at the DNA replication fork Figure** 6.13 of Hartl & Jones : Role of key proteins in DNA replication; draw 5’ and 3’ ends, leading, lagging strands

5. 21-5 Priming in Eukaryotes **Eukaryotic replication is more complex –Bigger size of eukaryotic genomes, most are linear –Slower movement of replicating forks –Each chromosome must have multiple origins Model monkey virus SV40 (5200 nt genome) Later yeast ARS (centromere) regions

6. Fig. 21.2 Replication of SV40 is bidirectional : Isolate replicating molecules; cleave with EcoRI that has 1 site; look at molecules in EM (A-j = increasing replication).

7. 21-7 Origin of Replication in SV40 SV40 ori adjacent to transcription control region Initiation of replication needs viral large T antigen (major product of early transciption) binding to: –Region within 64-bp ori core –Two adjacent sites T antigen helicase activity opens up replication bubble within ori core Priming carried out by primase associated with host DNA pol 

8. Fig. 21.4 Point mutations define critical regions of SV40 ori: AT regions; T-Ag binding site < -Early genesLate genes ->

9. 21-9 Autonomously replicating sequences (ARSs) 4 important regions: –Region A - 15 bp long with11-bp consensus sequence highly conserved in ARSs –B1 and B2 –B3 may permits important DNA bend within ARS1 ARS is Yeast Origin of Replication permit replication of gene in yeast linker scanning mutants define critical regions: plasmid has centromere, URA gene; grow non-selective and then check Ura+ (Fig. 7)

10. 21-10 21.2 Elongation and processivity Once primer in place,DNA synthesis begins Coordinated synthesis of lagging and leading strands keeps pol III holoenzyme on template Replication is highly processive, very rapid: pol III holoenzyme in vitro ~ 730 nt/sec (in vivo ~ 1000 nt/sec) Pol III core alone is poor polymerase: after ~10 nt it falls off Takes time to reassociate with template and nascent DNA Missing from core enzyme is processivity factor: –‘ sliding clamp ’,  -subunit of holoenzyme (see Table 20.2)

11. 21-11 Processivity agent:  -Subunit is clamp; keeps pol III on DNA Core plus  -subunit replicates DNA processively –(~ 1,000 nt/sec) –Dimer formed by  -subunit is ring-shaped –Ring fits around DNA template –Interacts with  -subunit of core to tether whole polymerase and template (Fig. 9) Holoenzyme stays on template with  -clamp (Fig. 11) Fig. 12  -dimer on DNA

12. 20-12 Pol III Holoenzyme Table 20.2 Pol III core has 3 subunits; Pol III  complex has 5 subunits – DNA-dependent ATPase Pol III holoenzyme includes  subunit

13. Fig. 21.10 DNA pol III subunits bind each other:  core;  ATPase,  clamp Purified subunits mixed and chromatographed to separate complexes from free proteins SDS-PAGE + Western blot tests which proteins in which complexes Also assayed DNA polymerase activity

14. Eukaryotic processivity factor PCNA forms trimer, a ring that encircles DNA and holds DNA polymerase on the template 21-14 Fig. 14 Fig. 13  -dimer on DNA in E. coli

15. 21-15  Clamp and  Clamp Loader  -subunit needs help from  complex to load onto DNA –This  complex acts catalytically to form processive  complex –  not remain associated with complex during processive replication Clamp loading is ATP-dependent –Energy from ATP changes conformation of loader so  -subunit binds one  -subunits –Binding opens clamp, allows it to encircle DNA

16. 21-16 Pol III* subassembly has 2 cores, one  and no  Fig. 17:  complex has 5 subunits Recall from table 2: Core pol III has 3 subunits:  is polymerase;  is exonuclease;  dimerizes core

17. 21-17 Simultaneous Strand Synthesis by double- headed pol III 2 core polymerases attached through 2  -subunits to  complex –One core responsible for continuous synthesis of leading strand –Other core performs discontinuous synthesis of the lagging strand –  complex serves as clamp loader to load  clamp onto primed DNA template –After loading,  clamp loses affinity for  complex; instead associates with core polymerase Fig. 18

18. 21-18 Lagging Strand Replication  complex and  clamp help core polymerase with processive synthesis of Okazaki fragment When fragment completed,  clamp loses affinity for core  clamp +  complex acts to unload clamp Now clamp recycles Fig. 25

19. 21-19 21.3 Termination of replication Straightforward for phage like that produce long, linear concatemers (rolling circle): Grows until genome-sized piece cut off, packaged into phage head Bacterial replication – 2 replication forks approach each other at terminus region –22-bp terminator sites bind specific proteins (terminus utilization substance, TUS) –Replicating forks enter terminus region, pause –2 daughter duplexes entangled, must separate Fig. 26

20. 21-20 Decatenation: Disentangling Daughter DNAs in Bacteria End of replication, circular bacterial chromosomes are catenanes: decatenated in 2 steps: –Melt unreplicated double-helical turns linking two strands –Repair synthesis fills in gaps –Decatenated by topoisomerase IV Fig. 27

21. 21-21 Eukaryotes have problem filling gaps left when RNA primers are removed after DNA replication: –DNA cannot be extended 3’  5’ direction –No 3’-end upstream (unlike circular bacterial chromosome) –If no resolution, DNA strands get shorter each replication Fig. 29 Termination in Eukaryotes: linear chromosomes role of telomerase

22. 21-22 Telomeres Telomeres - special structures at ends of chromosomes One strand of telomeres is tandem repeats of short, G-rich regions (sequence varies among species) –G-rich telomere strand is made by enzyme telomerase –Telomerase contains a short RNA that is template for telomere synthesis C-rich telomere strand is synthesized by ordinary RNA-primed DNA synthesis –Process like lagging strand DNA replication Ensures chromosome ends are rebuilt, do not suffer shortening each round of replication

23. Fig. 21.30 Tetrahymena cells have telomerase activity: Greider & Blackburn Cell extracts, synthetic oligo; + 32P-dNTPs, other dNTP Conclusion: enzyme adds 6-bp units Only needs GTP, TTP (lanes 3, 6) template (TTGGGG) 4

24. 21-24 Telomere sequences vary: Tetrahymena: TTGGGG Vertebrates: TTAGGG Yeast: TTGGG Fig. 31 Telomere Formation: telomerase makes DNA from RNA template: TERT, telomerase reverse transcriptase: proteins p43 and p123 1 RNA template Telomerase activity is high: in normal cells S phase, in cancer cells always

25. 21-25 Telomere Structure Eukaryotes protect telomeres from nucleases and ds break repair enzymes Ciliates have TEBP (telomere end-binding protein) to bind and protect 3’-single-strand telomeric overhang Budding yeast has Cdc13p which recruits Stn1p and Ten1p that all bind ss telomeric DNA Mammals and fission yeast have protein similar to TEBP binding to ss telomeric DNA Fig. 32

26. 21-26 Mammalian Telomeres T loop protects ss telomeric DNA (G-rich 3’ end loops) Proteins TRF1 and TRF2 help telomeric DNA form loop in which ss 3’-end of telomere invades ds telomeric DNA TRF1 may bend DNA into shape for strand invasion TRF2 binds at point of strand invasion, may stabilize displacement loop Fig. 36

27. Review questions 2. List the components of E. coli primosome and roles in primer synthesis. 4. Outline strategy for identify yeast ARS sequence. 14. How can discontinuous synthesis of lagging strand keep up with continuous synthesis of leading strand? 21. Why do eukaryotes need telomeres, but prokaryotes do not? 21-27