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