pnas, july 5, 2006. vol 103, no 27. the cell cycle r. sears, con 664 lecture
TRANSCRIPT
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PNAS, July 5, 2006. vol 103, no 27
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The Cell Cycle
R. Sears, Con 664 lecture
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DNA Replication Initiation
Pre-replicative complex assembly
• Initiated during M/G1 transition
• Maintained throughout M phase
• Regulated by CDK activity
Int Rev Cytol. 2007;256:69-109
Geminin
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Pre-RC Components of CMG
• Cdc45• biochemical function undetermined• tightly associates with Sld3 and the GINS tetramer• critcal component for DNA unwinding at the origin.
• Mcm2-7• minichromosomal maintenance proteins 2, 3, 4, 5, 6, and 7• forms DNA binding hexameric ring• contains elusive helicase activity• Purified Mcm 4, 6, and 7 show ATPase activity only when not bound to Mcm 2, 3, or 5 (Schwacha and Bell. Mol Cell. 2001 Nov;8(5):1093-104)
• GINS• heterotetramer of Sld5, Psf1, Psf2, Psf3 (Go, Ichi, Ni, San)• cricital for association of Cdc45 to pre-RC• Thought to promote association of Pol ε via Dpb11
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“Mcm Paradox”• Stoichiometry of Mcm proteins are much greater than
number of replication forks. (J Biol Chem. 2002 Sep 6;277(36):33049-57)
– 10 to 40 Mcm2-7 complexes load per replication fork– loading is ORC dependent, but distribute to a large region
surrounding ORC.
• Furthermore, Mcm proteins don’t preferentially localize to sites of DNA replication (J Cell Sci. 1996 Feb;109 ( Pt 2):309-18)
– Mcm proteins Cdc21, Cdc46, Mcm3 bind unreplicated chromatin– Displaced from replicating chromatin– Serves as a marker for unreplicated DNA
• F1-ATPase like activity- “rotary pump model” (EMBO Rep. 2003 Jan;4(1):26-30)
– distributed Mcm hexamers translocate DNA toward rep fork
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EMBO Rep. 2003 Jan;4(1):26-30
Rotary pumping model
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Short intro to Helicases(adaptation courtesy of Dr. Hoatlin)
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Helicases
• Molecular motors using ATP hydrolysis to catalyze the unwinding of DNA duplex
• Roles in replication, repair, recombination, transcription
• Move unidirectionally along DNA
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Helicase Terminology
• Motor along DNA using ATP hydrolysis to unwind
• 2004: All helicases are translocases and DNA-dependent ATPases
• Short motifs, termed Q (new), Ia, Ib, II, III, IV, V and VI (DEAD/H family)
• MCMs• RecQ (Sgs1 in yeast)
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Structure of a DNA Helicase
Hexamer in a ring form
Replication fork and helicase to scale
Bacteriophage T7 replicative helicase (red is ATP). Six identical s.u. bind single strand and duplex DNA alternately and hydrolyze ATP in an ordered fashion to propel molecule along DNA-- a single strand that passes through the central hole…it ripples
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Helicase Needs
• Usually require ssDNA loading zone-binding seq independent
• Exceptions are RecBCD and LTag, RuvB: all like ds DNA
• Many need a replication fork like structure• Some (RecBCD,UvrD, Rep, RecQ, LTag) can
unwind from blunt ended duplex DNA• E. coli RecG, RuvA, and RuvB recognize
Holliday junctions
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Tuteja, Narendra & Tuteja, Renu (2004)European Journal of Biochemistry 271 (10), 1835-1848.
Helicase Polarity
Substrates used to determine direction of unwindingIf 3’-5’ direction suggests leading strand placement*5 in the figure is the labeled end and is released if substrate is unwound
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Tuteja, Narendra & Tuteja, Renu (2004)European Journal of Biochemistry 271 (10), 1835-1848.
Unwound
Substrate
General Unwinding Assay
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Crystal Structure of Human GINS
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Each monomer has structural similarity
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Crystal Structure of Human GINS
1. Novel quaternary arrangement, not at all like PCNA
2. Novel B-domain folds identified
3. Overall highly acidic surface
4. No good clefts for binding DNA
5. Protomer interfaces are very tight and hydrophobic- not likely to undergo conformational changes.
6. Psuedo-two fold symmetry in tetramer suggests evolutionary divergence from archeal GINS (α2β2 tetramer)
7. Conserved surface on Psf1 subunit is a probable location for Cdc45 or MCM binding
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Why Xenopus egg extracts?
breifly reviewed in: Nature Protocols 1, 2305 - 2314 (2006)
• Easy to mass produce– Frogs can be horomonally stimulated to spawn frequently
• Synchronized cell cycle– All eggs are arrested at G2/M in metaphase of meosis– Division and replication machinery poised to fire
• Protein rich– Abundant in maternally transcribed protein
• Easy to manipulate– eggs maintained in arrest by calcium chelators (EGTA)– induced to complete the cell cycle by adding Ca++
– Cell cycle reset back to G2/M by adding fresh extract (CSF)– Extracts can be selectively immunodepleted, or reagents added
to specific concentrations
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Purification Scheme
DEAE Sepharose
Superdex-200
Poros-Heparin
Mono S
Mono Q
Anti Cdc45 IP
50% AS ppt
Weak anion exchanger- CMG binds at 100 mM KCl elutes at 380 mM KCl
SEC good for large proteins and complexesexchanges buffer back to 100 mM KCl
Highly sulfated sugar polymer. Used for cation exchange chromatography. CMG elutes at 190-265 mM KCl
Cation exchanger, used here as a “negative” purification step, CMG does not bind.
Quaternary Amine based anion exchangerCMG binds at 60 mM KCl, elutes at 340-400 mM KCl
Two methods: IP with polyclonal α-Cdc45, elute at pH 2.5IP with “peptide B” purified α-Cdc45, elute at neutral pH with peptide B
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Supp Fig 9
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Significance of the work
Currently, only indirect evidence for Mcm2-7 as the main replicative helicase– required for replication initiation and elongation– required for DNA unwinding– sequence similarity to AAA+ family proteins– archael homologs show robust helicase activity– eukaryotic homologs show weak helicase activity for
Mcm4, 6, and 7 only
This paper shows strong evidence for replicative helicase activity in Mcm2-7, and that its function is dependent on binding to Cdc45 and GINS
This CMG complex is thought to form the core helicase activity of the “replisome progression complex”