initiation of dna replication lecture 3oge.med.ufl.edu/courses/gms 6001/bloom_l03...
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1!
Initiation of DNA ReplicationLecture 3
!
Linda Bloom!Office: ARB R3-165!
email: [email protected]!phone: 392-8708!
Lecture 3 Outline!• Students will learn!
– Basic techniques for visualizing replicating DNA!– What a replication origin is.!– How replication is initiated at origins.!– Regulation of initiation.!
• Students should know!– Basics of DNA structure!– Basic replication mechanisms (previous 2 lectures0!
• Reading Assignment!– Lodish - Chp. 19.4 pp. 890-897!– Voet & Voet – Chp. 30.3Ca & b, pp 1194 – 1196;
30.4A, pp 1201-1202; 30.4Bb, c, d, pp 1205 - 1206!2!
3!
The Replicon Hypothesis!
• Two basic elements required to initiate DNA replication!– François Jacob, Sydney Brenner & Jacques Cuzin (1963)!
– replicator = a cis-acting factor!• a genetic element required or sufficient to direct initiation of replication!• origin of replication - specific sites at which DNA unwinding and
replication begin!• E. coli = OriC!• S. cerevisiae = ARS!
– initiator = a trans-acting factor!• a protein that specifically recognizes DNA element(s) in the replicator!• binds origin and recruits other replication proteins!• E. coli = DnaA!• eukaryotes = ORC!
– replicon!• unit of DNA that is replicated under the control of a replicator/initiator!
4!
Replication of the E. coli Genome!
OriC!
≈ 5 x 106 bp!
bidirection synthesis!
�theta� (θ) structure!
fork movement!500 - 1000 nt/s!
the E. coli genome contains!a single replicon!
5!
Replication of Eukaryotic Genomes!
bidirectional synthesis!
fork progression typically!10X slower than bacteria!≈ 50 - 100 nt/s!
multiple origins!
human genome estimated 30,000 replicons,!average size around 100 kb!
Drosophila Replication Bubble with 2 Forks!
Autoradiogram of E. coli Chromosome!
• Bacteria were grown in medium containing [3H]thymidine!• Radiolabeled thymidine was incorporated into DNA!• DNA isolated, stretched on a slide, and exposed to film!
Visualization of Origin-dependent DNA Replication – A �Classic� Method, DNA Fiber Autoradiography!
• Synchronize cells (later days)!
• Deplete dTTP by treating with 5-FUdR!
• Pulse with 3H-thymidine and chase with cold!
• Vary pulse and chase times in different expts!
• Isolate DNA by filter binding!
• Expose to film!
8!
DNA Fiber Autoradiography – Real Data!
9!
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PLATE IV. Tandem arrays of autoradiograms produced in a pulse-chase experiment. Labeling
and autoradiographg were performed as in the legend to Plate III. Dark field. J.A. Huberman & A.D. Riggs (1968) On the mechanism of replication in mammalian chromosomes. J. Mol. Biol. 32, 327-341.!
CHO Cells!
DNA Fiber Fluorography
• Uses bromo-, chloro-, and iododeoxyuridine (BrdU, CldU, and IdU) visualized by immunofluorescence
– Also other dNTP�s – biotin-labeled, digoxigenin-labeled
• Can synchronize cells, or use two �colors� with asynchronous cells
• DNA stretched onto glass surface such as slide coverslips
• Coupled to FISH (fluorescence in situ hybridization) to identify specific DNA regions or sequence
10!
�Two color� Pulse-labeling in Asynchronous Cells
11!
• Cells are not synchronized
• Pulse with IdU for a specified period of time
• Followed by a pulse with CldU for a defined period of time
C. Conti, J.A. Seiler, and Y. Pommier, Cell Cycle 6, 2760-2766 (2007)!
12!
Identification of Origins of Replication!
1) insert a segment of genomic DNA into a plasmid with a selectable marker!
2) Put plasmids in cells!
In prokaryotes, some viruses, and yeasts, replicators (origins) were identified by their ability to support replication of extrachromasomal DNA (a plasmid)!
4) Isolate cells that can grow on selection media!
3) Grow cells!
13!
E. coli Origin of Replication, OriC!
• oriC = 245 bp!– 5 DnaA boxes = �strong� boxes!
• 9mer sequence elements!• 5�TT(A/T)TNCACA!
– AT-rich sequences!• 3 x 13mer!• 5' G A T C T n T T n T T T T 3'!
– 11 GATC sites!
W. Messer, FEMS Microbiology Reviews 26, 355-374 (2002)!
recognition!
DNA unwinding element!(DUE)!
Structures of Yeast Origins!
14!from D. M. Gilbert (2001) Science 294, 96 - 100!
But, Initiation of Replication in Eukaryotes is More Complicated than in Bacteria!
• Although the replicon model was presumed for eukaryotes and initially seemed valid because of identification of origins in yeast, it quickly became clear that initiation of replication was much more complicated!
• Many origins on a single chromosome!• Different origins used a different stages of development!• Origin usage different under different growth conditions & tissues!• Origins �fire� at different times during S-phase!• Many origins never fire!• The replicator is unlikely to be defined by a specific consensus-
sequence!– Even in S. cerevisiae that has consensus ARS sequences not all are
active origins!
15!
E. coli Origin of Replication, OriC!
Initiation of Replication in E. coli!
Identification of the Eukaryotic Initiator!
• ORC = Origin Recognition Complex!
• S.P. Bell and B. Stillman (1992) ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128 – 134.!
• Used the S. cerevisiae ARS1 to purify a DNA binding complex from yeast nuclear extracts!
– Fractionated extracts using different procedures including column chromatography!
– Assayed fractions for ORC binding activity!
– Optimized purification included S-sepharose, Q-sepharose, and DNA (A sequence from ARS1) affinity chromatography!
• ARS1 binding was ATP-dependent!
– Used DNA footprinting to characterize binding!
18!
19!
ORC is the initiator in eukaryotes!
• a six-subunit complex = Orc1 - 6!• Orc1- Orc5 are members of the AAA+ family of ATPases!• replicator identified in yeast & used to find initiator!• in higher eukaryotes, initiator identified by homology!
20!
Initiation of DNA Replication is Tightly Regulated In Eukaryotes!
• Goal to replicate each chromosome once and only once per cell cycle!– Don�t want to enter mitosis with too many or too few chromosomes, or
incompletely replicated chromosomes!
• Initiation occurs in two phases!– Replicator selection!
• occurs in G1 phase!• formation of pre-RC�s!
– pre-RC = pre-replicative complex!
– Origin activation!• occurs in S phase!• requires activity of cell-cycle-dependent kinases!
– Cdk = cyclin dependent kinases!• formation of pre-initiation complexes!
21!
Cell Cycle Regulation of Pre-RC Formation!
Fig. 8-30. Molecular Biology of the Gene, 5th Ed., Watson, J.D. et al.!
Cdc7 & Cdk2!
22!
Eukayrotic Origins are Replicated Only Once!
Fig. 8-28. Molecular Biology of the Gene, 5th Ed., Watson, J.D. et al.!
23!
Regulation of Initiation by Cyclin-dependent Kinases!
Fig. 8-30. Molecular Biology of the Gene, 5th Ed., Watson, J.D. et al.!
24!
Cell Cycle Regulation of Pre-RC Formation!
Fig. 8-30. Molecular Biology of the Gene, 5th Ed., Watson, J.D. et al.!
Cdc7 & Cdk2!