New histone supply regulates replication fork speed and PCNA unloading

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.     

体外装配核小体过程组蛋白浓度依赖的动力学模型

为探索组蛋白浓度对核小体体外装配的影响,本研究表达纯化了4种组蛋白,通过控制实验反应体系中组蛋白的浓度,利用盐透析法在体外装配了核小体,检测分析了组蛋白浓度与核小体组装效率的关系。以此实验数据为基础,提出了核小体组装过程组蛋白浓度依赖性的动力学模型。实验结果发现,反应体系中组蛋白浓度与核小体生成量呈典型的线性关系。依据动力学理论模型,进行线性回归拟合,回归系数达到0.963;经计算601 DNA序列组装核小体的反应速率常数k为1.49×10^-5mL·h·μg^-1。CS1序列验证动力学模型的线性回归相关系数为0.989,反应速率常数为1.52×10^-5mL·h·μg^-1。该实验方法及动力学模型中反应速率常数k可用于评价相同长度的DNA序列组装核小体的能力、组蛋白与其突变体以及组蛋白变体之间形成核小体结构能力的差异。该动力学模型的建立为理解核小体装配、核小体定位、染色质结构等相关问题提供了理论指导。     

Regulation of histone synthesis and nucleosome assembly

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.     

Temporal dynamics of cytomegalovirus chromatin assembly in productively infected human cells

The genomes of herpesviruses, including human cytomegalovirus (CMV), are double-stranded DNA molecules maintained as episomes during infection. The viral DNA lacks histones when encapsidated in the virion. However, it has been found histone associated inside infected cells, implying unidentified chromatin assembly mechanisms. Our results indicate that components of the host cell nucleosome deposition machinery target intranuclear CMV DNA, resulting in stepwise viral-chromatin assembly. CMV genomes undergo limited histone association and nucleosome assembly as early as 30 min after infection via DNA replication-independent mechanisms. Low average viral-genome chromatinization is maintained throughout the early stages of infection. The late phase of infection is characterized by a striking increase in average histone occupancy coupled with the process of viral-DNA replication. While the initial chromatinization affected all analyzed parts of the CMV chromosome, a subset of viral genomic regions, including the major immediate-early promoter, proved to be largely resistant to replication-dependent histone deposition. Finally, our results predict the likely requirement for an unanticipated chromatin disassembly process that enables packaging of histone-free DNA into progeny capsids.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The process of coordinated DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3–H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three his-tone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3–H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3–H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.Key words: Gen5, Rtt109, chromatin, nucleosome assembly, genome integrity     

Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding

Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication. Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position. Histone exchange as well as nucleosome sliding is independent of ATP and relies on the presence of the C-terminal acidic domain of yeast NAP-1, even though this region is not required for histone binding and chromatin assembly. Our results suggest a novel role for NAP-1 (and perhaps other acidic histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding. NAP-1 may function either untargeted (if acting alone) or may be targeted to specific regions within the genome through interactions with additional factors.     

Stability of the conservative mode of nucleosome assembly.

The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.     

The ins and outs of nucleosome assembly

De novo nucleosome assembly coupled to DNA replication and repair in vitro involves the histone chaperone chromatin assembly factor 1 (CAF-1). Recent studies support a model in which CAF-1 can be targeted to newly synthesized DNA through a direct interaction with proliferating cell nuclear antigen (PCNA) and can act synergistically with a newly identified histone chaperone. Insights have also been obtained into mechanisms by which this CAF-1-dependent pathway can establish a repressed chromatin state.     

Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins.

Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood. We demonstrate that chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication. Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro. We suggest that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase. Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation. The HP1 proteins reassociate with chromatin at the end of mitosis, as histone H3 is dephosphorylated.