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DNA Replication (III)

DNA Replication (III). 王之仰. Two replication forks might assemble at a single origin and then move in opposite directions, leading to bidirectional growth of both daughter strands.

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DNA Replication (III)

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  1. DNA Replication (III) 王之仰

  2. Two replication forks might assemble at a single origin and then move in opposite directions, leading to bidirectional growth of both daughter strands. • In the case of SV40 DNA, replication is initiated by binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins to form two replication forks.

  3. These then move away from the SV40 origin in opposite directions with leading- and lagging- strand synthesis occurring at both forks. • Unlike SV40 DNA, eukaryotic chromosomal DNA molecules contain multiple replication origins separated by tens to hundreds of kilobases.

  4. A six-subunit protein called ORC, for origin recognition complex, binds to each origin and associates with other proteins required to load cellular hexameric helicases composed of six homologous MCM proteins. • Two opposed MCM helicases separate the parental strands at an origin, with RPA proteins binding to the resulting

  5. single-stranded DNA. Synthesis of primers and subsequent steps in replication of cellular DNA are thought to be analogous to those in SV40 DNA replication. • Replication of cellular DNA and other events are tightly regulated, so the appropriate numbers of cells for each

  6. tissue are tightly regulated, so that the appropriate numbers of cells are produced during development and throughout the life of an organism. • Control of the initiation step is the primary mechanism for regulating cellular DNA replication.

  7. Activation of MCM helicase activity, which is required to initiate cellular DNA replication, is regulated by specific protein kinases called S-phase cyclin-dependent kinases. • Other cyclin-dependent kinases regulate additional aspects of cell proliferation, including two daughter cells.

  8. E. coli DNA polymerases introduces about 1 incorrect nucleotide per 104 polymerized nucleotides. • Proofreading depends on a 3’ →5’ exonuclease activity of DNA polymerases. • When an incorrect base is incorporated during DNA synthesis, base-pairing between the 3’ nucleotide of the nascent

  9. strand and the template strand does not occur. • The polymerase pauses, then transfers the 3’ end of the growing chain to its exonuclease site, where the incorrect mispaired base is removed. Then the 3’ end is transferred back to the polymerase site, where this region is copied correctly.

  10. Two eukaryotic DNA polymerase δ and ε, used for replication of most chromosomal DNA in animal cells, also have proofreading activity. • It seems likely that proofreading is indispensible for all cells to avoid excessive mutations.

  11. Eukaryotic chromosomes are replicated from multiple replication origins. • Initiation of replication from these origins occurs throughout S phase. • No eukaryotic origin initiates more than once per S phase. • The S phase continues until replication

  12. from multiple origins along the length of each chromosome results in complete replication of the entire chromosome. • These two factors ensure that the correct gene copy number is maintained as cells proliferate. • Yeast replication origins contain an 11

  13. -base-pair conserved core sequence to which is bound a hexameric protein, the origin-recognition complex (ORC), required for initiation of DNA synthesis. • The ORC remains associated with origins during all phases of the cycle. • Several additional replication initiation factors were required to initiate DNA synthesis.

  14. These DNA replication initiation factors associate with the ORC at origins during G1; During G1 the various initiation factors assemble with the ORC into a prereplication complex at each origin. • The restriction of origin “firing” to once and only once per cell cycle in S. cerevisiae is enforced by the alternating cycle of B-type cyclin-CDK activity levels

  15. through the cell cycle: low in telophase through G1 and high in S, G2, and M through anaphase. • S-phase cyclin-CDK complexes become active at the beginning of S phase when their specific inhibitor, Sic1, is degraded. • The prereplication complexes assembled at origins early in G1 initiate DNA synthesis in S phase when they are

  16. phosphorylated by the S-phase cyclin-CDKs and a second heterodimeric protein kinase, DDK, expressed in G1 along with other proteins involved in DNA replication. • At least one subunit of the hexameric MCM helicase and Cdc6 is required; Following their phosphorylation, the helicase unwinds the DNA, and the resulting single-stranded DNA is bound by the single-stranded binding protein RPA and

  17. other replication factors. • As the replication forks progress away from each origin, the phosphorylated initiation factors are displaced from the chromatin. • ORC complexes immediately bind to the origin sequence in the replicated daughter duplex DNA and remain bound throughout the cell cycle.

  18. Origins can fire only once during the S phase because the phosphorylated initiation factors cannot reassemble into a prereplication complex. • Phosphorylation of components of the prereplication complex by S-phase cyclin-CDK complexes and the DDK complex simultaneously activates initiation of DNA

  19. replication at an origin and inhibits re-initiation of replication at that origin. • B-type cyclin-CDK complexes remain active throughout the S-phase, G2, and early anaphase, maintaining the phosphorylated state of the replication initiation factors that prevents the

  20. assembly of new prereplication complexes. • When the Cdc14 phosphatase is activated in late anaphase and the APC/C-Cdh1 complex triggers degradation of all B-type cyclins in telophase, phosphates on the initiation

  21. factors are removed by the unopposed Cdc14 phosphatase; This allows the reassembly of prereplication complexes during early G1. • The inhibition of APC/C activity in G1 sets the stage for accumulation of the S-phase cyclins needed for onset of the

  22. next S phase. • This regulatory mechanism has two consequences: (1) prereplication complexes are assembled only during G1, when the activity of B-type cyclin-CDK complexes is low; (2) each origin initiates replication

  23. one time only during the S phase, when S phase cyclin-CDK complex activity is high.

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