1. Control of DNA replication Replicon Origins and terminators Solutions to the “end problem” (telomeres) Cellular control mechanisms

2. 3 stages to replication Initiation: begin at a specific site, e.g. oriC for E. coli. Elongation: movement of the replication fork Termination: at ter sites for E. coli

3. Replicon = unit that controls replication Replicator: cis-acting DNA sequence required for initiation; defined genetically Origin: site at which DNA replication initiates; defined biochemically Initiator: protein needed for initiation, acts in trans

4. Replication “eyes”

5. Theta-form replication intermediates visualized in EM for polyoma virus B. Hirt

6. Bidirectional and unidirectional replication

7. For completed molecules, label appears first in the fragments of DNA synthesized last

8. Labeling of completed DNA molecules can map replication origins Dana and Natahans, 1972, PNAS: map the replication origin of SV40 by labeling replicating molecules for increasing periods of time, isolating complete molecules, digesting with Hind restriction endonucleases, and determining which fragments have the most radioactivity.

9. Data from labeling completed DNAs Relative amount of pulse label Fragment5 min10 min15 min A1.01.01.0 B3.93.02.3 C00.750.75 D0.920.861.1 E1.82.01.7 F4.03.12.4 G5.44.22.6 H1.72.52.0 I2.73.02.2 J4.93.72.6 K2.42.91.9

10. Position of ori for SV40

11. Replicating molecules have different shapes generated by replication bubbles and forks

12. Bubble arcs on 2-D gels A fragment containing an origin will have one or two replication forks moving through it, generating bubbles of increasing size. These will be detected as bubble arcs on 2-D gels of the replicating DNA, when that region is used as a hybridization probe. 1st dimension: size 2nd dimension: size and SHAPE Unit length Twice unit length

13. Y-arcs on 2-D gels of replicating molecules A replication fork moving through a region will show a Y-arc on 2-D gels of the replicating DNA, when that region is used as a hybridization probe. 1st dimension: separate by size 2nd dimension: separate by size and SHAPE Brewer and Fangman, 1987

14. 2-D gels: map number & position of replication origins

15. Example of analysis of a replicon using 2-D gels Restriction fragments: Fork movement origin termination

16. Positions of oriC and ter in E. coli Replication fork 2

17. Features of oriC oriC was identified by its ability to confer autonomous replication on a DNA molecule, thus it is a replicator. Studies show that chromosomal DNA synthesis initiates at oriC, thus it is also an origin of replication. Replication from oriC is bidirectional.

18. Structure of oriC 245 bp long –4 copies of a 9 bp repeat –3 copies of a 13 bp repeat –11 GATC motifs 13 9999 1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA 121 AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA TCTCTTATTA GGATCGCACT 181 GCCCTGTGGA TAACAAGGAT CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT 241 GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA GAATGAGGGG TTATACACAA 301 CTCAAAAACT GAACAACAGT TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC 361 AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT TGAGTAAATT AACCCACGAT

19. Conservation of oriC in enteric bacteria

20. Proteins needed for initiation at oriC #1 DnaA –Only used at initiation –Mutations cause a slow-stop phenotype –Binds to the 4 copies of 9 bp repeats –Further cooperative binding brings in 20 to 40 DnaA monomers –Melts the DNA at the 3- 13 bp repeats

21. DnaB –ATP-dependent helicase –Displaces DnaA and unwinds DNA further to form replication forks –“Activates” primase, apparently by stablizing a secondary structure in single-stranded DNA DnaC –Is in complex with DnaB before loading onto template Proteins needed for initiation at oriC #2

22. DnaG primase Gyrase SSB All but DnaA are also used in elongation Proteins needed for initiation at oriC #3

23. Initiation at oriC: Model

24. Positions of ter sequences in E. coli Replication fork 2

25. Termination of replication: DNA sites and proteins needed DNA sites: ter sequences, 23 bp –terD and terA block progress of counter- clockwise fork, allow clockwise fork to pass –terC and terB block progress of clockwise fork, allow counter- clockwise fork to pass Protein: Tus –“ter utilization substance” –Binds to ter –Prevents helicase action from a specific replication fork

26. Termination and resolution

27. Control by methylation GATC motifs are substrates for methylation by dam methylase. Methylase transfers a methyl group from S- adenosylmethionine to N-6 of adenine in GATC. Methylated GATC on BOTH strands: oriC will serve as an origin Methylated GATC on ONLY one strand (hemimethylated): oriC is not active Re-methylation is slow, delays use of oriC to start another round of replication.

28. Regulation of replication by methylation G A T C C T A G m m G A T C C T A G m G A T C C T A G m G A T C C T A G m m G A T C C T A G m m replicate methylate (lags behind replication) Fully methylated Hemimethylated dammethylase Fully methylated Will replicate Will not replicateWill replicate

29. Replicores in E. coli

30. Correlation between genome structure and replication direction in E. coli Information from whole-genome sequencing : –Most genes are transcribed in the direction that the replication fork moves. –G>C on leading strand (“top strand” for replicore 1, opposite strand for replicore 2) –Oligonucleotides containing CTG are more frequent on the leading strand (i.e. template for lagging strand) in E. coli. These are the binding sites for DnaG (primase). –Recombination hotspot Chi: more abundant on leading strand