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PNAS, July 5, 2006. vol 103, no 27

PNAS, July 5, 2006. vol 103, no 27. The Cell Cycle. R. Sears, Con 664 lecture. DNA Replication Initiation. Geminin. Pre-replicative complex assembly Initiated during M/G1 transition Maintained throughout M phase Regulated by CDK activity. Int Rev Cytol. 2007;256:69-109.

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PNAS, July 5, 2006. vol 103, no 27

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  1. PNAS, July 5, 2006. vol 103, no 27

  2. The Cell Cycle R. Sears, Con 664 lecture

  3. DNA Replication Initiation Geminin • Pre-replicative complex assembly • Initiated during M/G1 transition • Maintained throughout M phase • Regulated by CDK activity Int Rev Cytol. 2007;256:69-109

  4. 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

  5. “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

  6. Rotary pumping model EMBO Rep. 2003 Jan;4(1):26-30

  7. Short intro to Helicases(adaptation courtesy of Dr. Hoatlin)

  8. Helicases • Molecular motors using ATP hydrolysis to catalyze the unwinding of DNA duplex • Roles in replication, repair, recombination, transcription • Move unidirectionally along DNA

  9. 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)

  10. Structure of a DNA Helicase Hexamer in a ring form 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 Replication fork and helicase to scale

  11. 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

  12. Helicase Polarity Substrates used to determine direction of unwinding If 3’-5’ direction suggests leading strand placement *5 in the figure is the labeled end and is released if substrate is unwound Tuteja, Narendra & Tuteja, Renu (2004) European Journal of Biochemistry271 (10), 1835-1848.

  13. General Unwinding Assay Substrate Unwound Tuteja, Narendra & Tuteja, Renu (2004) European Journal of Biochemistry271 (10), 1835-1848.

  14. Crystal Structure of Human GINS

  15. Each monomer has structural similarity

  16. Crystal Structure of Human GINS • Novel quaternary arrangement, not at all like PCNA • Novel B-domain folds identified • Overall highly acidic surface • No good clefts for binding DNA • Protomer interfaces are very tight and hydrophobic- not likely to undergo conformational changes. • Psuedo-two fold symmetry in tetramer suggests evolutionary divergence from archeal GINS (α2β2 tetramer) • Conserved surface on Psf1 subunit is a probable location for Cdc45 or MCM binding

  17. Why Xenopus egg extracts?breiflyreviewed 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

  18. Purification Scheme 50% AS ppt Weak anion exchanger- CMG binds at 100 mM KCl elutes at 380 mM KCl DEAE Sepharose SEC good for large proteins and complexes exchanges buffer back to 100 mM KCl Superdex-200 Highly sulfated sugar polymer. Used for cation exchange chromatography. CMG elutes at 190-265 mM KCl Poros-Heparin Cation exchanger, used here as a “negative” purification step, CMG does not bind. Mono S Quaternary Amine based anion exchanger CMG binds at 60 mM KCl, elutes at 340-400 mM KCl Mono Q Two methods: IP with polyclonal α-Cdc45, elute at pH 2.5 IP with “peptide B” purified α-Cdc45, elute at neutral pH with peptide B Anti Cdc45 IP

  19. Supp Fig 9

  20. 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”

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