哈佛大学分子生物学--DNA复制(第六讲).ppt

1、Transition to replication: from pre-RC to unwinding,MCM10 : essential function in the initiation of DNA replication in yeast. Required in Xenopus after the loading of Mcm2-7, before the loading of CDC45 CDC7/DBF4 : A DBF4 dependent kinase (DDK) which, together with Dbf4, is required for the initiati

2、on of DNA replication. Possible substrates of this kinase are the MCMs. How do we know this? Cdk/Cyclin: A protein kinase essential for the initiation of DNA replication. Substrates that are activated by Cdk for replication initiation are unclear. MCM, CDC6 and some ORC subunits are phosphorylated,

3、but this phosphorylation inactivates the proteins and prevents re-replication. CDC45 : it seems to act together with ORC and the MCMs to initiate DNA replication. Has a role in the loading of DNA primase/polymerase complex. It and the MCM proteins appear to move along with the replication fork.How d

4、o we know this?,Transition to Replication,S phase: Chromosome duplication,Mitosis: Chromosome segregation,Problems for the cell: (1) Alternation of S and M (2) Completion of S before M and vice versa,With their multiple origins, how does the eukaryotic cell know which origins have been already repli

5、cated and which still await replication? Two observations: When a cell in G1of the cell cycle is fused with a cell in S phase, the DNA of the G1 nucleus begins replicating early. Thus S phase cells have a positive factor for DNA replication that G1 cells lack. When a cell in G2 of the cell cycle is

6、fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again. Thus cells in G2 have a negative factor that disappears in

7、mitosis and/or lack a positive replication licensing factor until they pass through mitosis.,Control of Replication through the cell cycle,Cyclin/cdk protein kinase (i) yeast with temperature sensitive mutation in CDC28 (a cdk) arrest in G1 (ii) addition of a cdk inhibitor (e.g. p21) to Xenopus egg

8、extracts in G1 inhibits replication DBF4/CDC7 protein kinase (i) ts mutation of CDC7 in yeast blocks cell cycle in G1 (by-passed by a mutation in MCM5) (ii) antibody mediated depletion in Xenopus egg extracts,Positive factor present in S phase cells,CDC6 (i) degraded in yeasts after G1/S (ii) displa

9、ced out of the nucleus after G1/S in mammalian cells MCM2-7 (i) displaced out of the nucleus after G1/S in yeast (ii) loses tight association with chromatin after G1/S in mammalian cells CDC45 (i) loses tight association with chromatin after G1/S in mammalian cells Cdt1 (i) degraded in mammalian cel

10、ls during S phase. (ii) inactivated by interaction with a protein called geminin. ORC: may be inactivated by phosphorylation Mcm10: ?,Positive factor (replication licensing factor) absent in G2 cells, but present in G1 cells,HsCDC6,G1,S,Wild-type,Ser,Ala,Ser,Asp,Proper,Subcellular,Localizati,on of H

11、sCDC6 is Dependent on,Both Intact,Phosphorylation,Sites and a Functional Cy Motif,CDC6 in mammalian cells is exported out of the nucleus In S phase due to phosphorylation by cyclin/cdk,G1,S,GFP-MCM4 localization through the cell cycle in yeast,Movie: http:/users.ox.ac.uk/kearsey/mcm4.html,Highly act

12、ive cyclin/cdk protein kinase (i) inactivated in mitosis due to degradation of cyclins. (ii) artificial inactivation in G2 yeast with ts mutations in the cdk or by induction of a cdk inhibitor results in re-replication of DNA without mitosis. (iii) the nuclear export (in mammalian cells) or proteoly

13、sis (in yeast) of CDC6 is due to phosphorylation by cyclin/cdk. Therefore the negative factor present in G2 cells may also be responsible for the absence of a positive replication licensing factor in G2 nuclei! Geminin (i) elevated in G2 cells and degraded in mitosis (ii) addition of a stable form o

14、f the protein to Xenopus egg extract inhibits the loading of MCM2-7 on chromatin and prevents DNA replication (iii) interacts with Cdt1.,Negative factor present in G2 cells, but absent in G1 cells,Low cyclin/cdk activity is required at G1/S to initiate DNA replication Low cyclin/cdk activity in G1/S is inadequate to initiate mitosis High cyclin/cdk activity in G2 inhibits DNA replication High cyclin/cdk activity in G2 is required for mitosis,Solution to the alternation problem,Solution to the completion p