Replicación 2

1. Replicación 2 Regulación de la replicación

2. E. coli • oriC contains 11 repeats that are methylated on adenine on both strands. GATC CTAG Met Met

3. E. coli • Replication generates hemimethylated DNA, which cannot initiate replication. GATC GATC CTAG CTAG • There is a 13 min delay before the repeats are remethylated. Met Met

4. oriC contains 11 copies of GATC • A sequence of four residues is target for methylation at the N6 position of adenine by the Dam methylase.

5. Completely methylated origins are functional • The ability of a plasmid relying upon oriC to replicate in dam–E. coli depends on its state of methylation.

6. Completely methylated origins are functional • If the plasmid is methylated, it undergoes a single round of replication, and then the hemimethylated products accumulate. • So a hemimethylated origin cannot be used to initiate a replication cycle.

7. Two hypothesis • Initiation may require full methylation of the Dam target sites in the origin. Or • Initiation may be inhibited by hemimethylation of these sites.

8. Answer • The latter seems to be the case, because an origin of nonmethylated DNA can function effectively.

9. Hechos • Hemimethylated origins cannot initiate again until the Dam methylase has converted them into fully methylated origins. • The GATC sites at the origin remain hemimethylated for ~13 minutes after replication.

10. Metilación de sitios GATC • A typical GATC sites elsewhere in the genome, remethylation begins immediately (<1.5 min) following replication. • The promoter of the dnaA gene also shows a delay before remethylation begins. This long period is unusual, because at

11. The dnaA promoter is repressed while it is hemimethylated. • This causes a reduction in the level of DnaA protein. • So: • the origin itself is inert • The production of the crucial initiator protein is repressed.

12. Origins are sequestered after replication • SeqA binds to hemimethylated DNA and is required for delaying rereplication. • SeqA may interact with DnaA.

13. Origins are sequestered after replication • While the origins are hemimethylated, they bind to the cell membrane, and may be unavailable to methylases. • The nature of the connection between the origin and the membrane is still unclear.

14. Controlling reuse of origins is identified by mutations in the gene seqA. • The mutants reduce the delay in remethylation at both oriC and dnaA. • As a result, they initiate DNA replication too soon, thereby accumulating an excessive number of origins • This suggests that seqA is part of a negative regulatory circuit that prevents origins from being remethylated

15. Functions of SeqA. • SeqA binds to hemimethylated DNA more strongly than to fully methylated DNA. • It may initiate binding when the DNA becomes hemimethylated, and then its continued presence prevents formation of an open complex at the origin.

16. Functions of SeqA. • SeqA does not have specificity for the oriC sequence, and it seems likely that this is conferred by DnaA protein, which would explain genetic interactions between seqA and dnaA.

17. Oligomerization of the SeqA–N dimer. Guarné, A et al. Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization. The EMBO Journal (2005) 24, 1502–1511, doi:10.1038/sj.emboj.7600634

18. Oligomerization of the SeqA–N dimer. • (A) Ribbon diagram of a single SeqA–N subunit. Guarné, A et al. Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization. The EMBO Journal (2005) 24, 1502–1511, doi:10.1038/sj.emboj.7600634

19. Oligomerization of the SeqA–N dimer. • (B) A SeqA–N dimer. The two subunits are shown as yellow and green ribbon diagrams

20. Oligomerization of the SeqA–N dimer. • (C) The asymmetric unit contains two SeqA–N dimers related by a noncrystallographic dyad axis and a 43 screw axis. • The two SeqA–N dimers colored yellow–green and blue–red, respectively, are shown in a ribbons diagram (left) and molecular surface representation (right).

21. Oligomerization of the SeqA–N dimer. • (D) Two views of the SeqA–N filament. • The black bar indicates a complete helical turn consisting of four dimers. • The 43 axis and the noncrystallographic (gray arrows) and crystallographic (gray ovals) dyad axes are indicated.

22. Oligomerization of the SeqA–N dimer. • (E) Crystal packing of the SeqA filaments shown as a ribbon diagram. • Filaments pack according to the crystallographic 31 axis. • The central filament is shown with the SeqA monomers colored yellow and green. The top and bottom filaments are shown in light and dark gray, respectively.

23. A model of the interactions between a SeqA filament and DNA Guarné, A et al. Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization. The EMBO Journal (2005) 24, 1502–1511, doi:10.1038/sj.emboj.7600634

24. A model of the interactions between a SeqA filament and DNA • A SeqA–N dimer and a pair of crystallographic SeqA–C–DNA complexes are placed to share a common dyad axis. • The protein is shown as ribbon diagrams in dark (SeqA–N) and light (SeqA–C) blue and the DNA depicted as stick models. Guarné, A et al. Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization. The EMBO Journal (2005) 24, 1502–1511, doi:10.1038/sj.emboj.7600634

25. A model of the interactions between a SeqA filament and DNA • The full-length SeqA dimer–DNA model is allowed to multimerize according to the 43 screw axis of the SeqA–N filament. The four-fold screw axis is perpendicular to the plane. Each SeqA–N dimer and the SeqA–C pair bound to it are shown in dark and light shades of a distinct color. Space between the N- and C-terminal domains accounts for the flexible linker and avoids contacts or clashes between neighboring SeqA–C molecules related by the 43 screw axis. An orthogonal view placing the SeqA–DNA superhelix in plane is shown on the right panel. No artificial coordinates are introduced, and DNAs are left to be discontinuous between adjacent SeqA dimers. Guarné, A et al. Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization. The EMBO Journal (2005) 24, 1502–1511, doi:10.1038/sj.emboj.7600634

26. Model of SeqA sequestration and SeqA foci migration following the replication forks • (A) As replication initiates, SeqA binds newly generated hemimethylated GATC sequences within oriC, triggering origin sequestration

27. Model of SeqA sequestration and SeqA foci migration following the replication forks • (B) As the forks progress, more hemimethylated GATC sequences become available. Spacing between the newly occurring GATC sequences favors SeqA filament formation, which in turn favors cooperative binding of distant GATC sequences

28. Model of SeqA sequestration and SeqA foci migration following the replication forks • Only few SeqA molecules are required for origin sequestration and, therefore, oriC will remain hemimethylated while the intervening DNA sequences are remethylated.