DNA replication Semiconservative replication Replicative Structures Replication Fork.
Regulation of Replication Fork Progression Through Histone Supply and Demand
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Transcript of Regulation of Replication Fork Progression Through Histone Supply and Demand
Regulation of Replication Fork Progression Through Histone Supply and Demand
Anja Groth, et al.
Science 318, 1928 (2007)
• Associates with H3-H4
• Required for the nucleosomal assembly on the newly synthesized strand in-vitro
• Mediates nucleosomal disassembly for transcriptional activation of genes
Local Regulators of Histone Dynamics
Nap 1Nucleoplasmin
Nucleophosmin CAF 1 Hira
Rbap46/48 Set
Spt 6 Asf 1
• Identified in a screen for genes that, when overexpressed, cause a defect in silencing at the mating-type loci in S. cerevisiae
• Highly conserved among eukaryotes
• Regulates cell cycle
• Stabilizes the genome
• Involved in DNA damage and repair mechanisms
• Associates with H3-H4
• Required for the nucleosome assembly on the newly synthesized strand in-vitro
• Mediates nucleosome disassembly for transcriptional activation of genes
Antisilencing function 1 – Asf 1
Proteins Interacting with Asf 1
Asf 1 interacts with MCM2,4,6,7
H3-H4 bridges the interaction between Asf 1 and MCM 2,4,6,7
MCM Interaction with Asf 1
Asf 1 interacts with MCM 2,3,4,5,6,7
MCM Interaction with Asf 1
Effect of Asf 1 Depletion on Cell Cycle
Cell cycle gets delayed while replication factories are normal
ssDNA bound RPA
Helicase (MCM)
Asf 1 Depletion – Effect on MCM Function(An Indirect approach)
Functional Helicase → DNA Unwinds → RPA binds ssDNADefective Helicase → No DNA unwinding → No RPA on DNA
Asf 1 Depletion – Effect on MCM Function
Scanty RPA !Helicase function of MCM 2-7 might be defective
Asf 1 Depletion – Effect on MCM Function(Polymerase – Helicase uncoupling: Direct approach)
Functional Helicase → DNA Unwinds → Antibody binds BrdU labelled ssDNADefective Helicase→No DNA unwinding→Antibody can’t bind Brdu labelled dsDNA
RPA
Asf 1 Depletion – Effect on MCM Function
BrdU
Helicase function of MCM 2-7 is defective
PCNA ssDNA Merge
Polymerase – MCM uncoupling:Effect on Asf 1 interaction with MCM 2-7
Asf 1 - (H3-H4) – MCM could be an intermediate in parental histone transfer
Effect of H3 - H4 Over Expression
H3-H4 Over expression arrested majority of cells in late S/G2 phase mimicking the Asf 1 depletion
24 hr -Tet treatment
Effect of H3 - H4 Over Expression
Effect of H3 - H4 Over Expression
Ectopic expression of Asf 1 alleviated the response to H3-H4 over expression
Depletion of Asf 1 aggavated the response to H3-H4 over expression
Regulation of Replication Fork Progression Through Histone Supply and Demand
Anja Groth, et al.
Science 318, 1928 (2007)
• Identified in a screen for genes that, when overexpressed, cause a derepress silencing at the telomeres in S. cerevisiae
• Highly conserved among eukaryotes
• Regulates cell cycle
• Stabilizes the genome
• Involved in DNA damage and repair mechanisms
• Associates with H3-H4
• Required for the nucleosome assembly on the newly synthesized strand in-vitro
• Mediates nucleosome disassembly for transcriptional activation of genes
Antisilencing function 1 – Asf 1
Copyright © 2007 by the Genetics Society of America
Singer, M. S. et al. Genetics 1998;150:613-632
Figure 1. Overexpressed cDNA genes disrupt silencing of telomeric URA3 and ADE2
(A) URA3 expression was measured in terms of the viability of the strains on medium lacking uracil. Five independent transformants of each clone were tested, and the median value for viability in the absence of uracil is designated by the height of each bar. Each error bar indicates the difference between the median and the maximum value. (B) ADE2 expression was reflected by the proportion of white (ADE2 on) vs. red (ADE2 off) sectors in the colonies of each strain.
Accumulation of asf1 and asf1 cac1 mutant yeast strains in G2/M. DNA content of asynchronous cultures of strains determined by flow
cytometric analysis.
H3-H4 bridges the interaction between Asf 1 and MCM 2,4,6,7
MCM Interaction with Asf 1
Asf 1a wt = 22.96745 kDaAsf 1b = 22.43236 kDaAsf 1a V94R = 23.02450 kDa
Effect of Asf 1 Depletion on Cell Cycle
Cell cycle gets delayed while replication factories are normal