Activation Of DNA Replication Checkpoint Research Articles

Mrc1 regulates parental histone segregation and heterochromatin inheritance

Chromatin-based epigenetic memory relies on the symmetric distribution of parental histones to newly synthesized daughter DNA strands, aided by histone chaperones within the DNA replication machinery. However, the mechanism of parental histone transfer remains elusive. Here, we reveal that in fission yeast, the replisome protein Mrc1 plays a crucial role in promoting the transfer of parental histone H3-H4 to the lagging strand, ensuring proper heterochromatin inheritance. In addition, Mrc1 facilitates the interaction between Mcm2 and DNA polymerase alpha, two histone-binding proteins critical for parental histone transfer. Furthermore, Mrc1's involvement in parental histone transfer and epigenetic inheritance is independent of its known functions in DNA replication checkpoint activation and replisome speed control. Instead, Mrc1 interacts with Mcm2 outside of its histone-binding region, creating a physical barrier to separate parental histone transfer pathways. These findings unveil Mrc1 as a key player within the replisome, coordinating parental histone segregation to regulate epigenetic inheritance.

Abstract 3174: Crosstalk between protein translation and DNA replication during stress response and cancer therapy

Abstract Tumor microenvironment, which includes both various types of cells (tumor cells, stromal cells, immune cells, etc.) and an interstitial fluid system, plays a key role in tumorigenesis, therapy response, and disease outcome. While the cell component has been heavily studied in cancer treatment, the interstitial fluid system has not. A key feature of the fluid system is constant oscillation of osmolytes such as salt, glucose, and proteins due to unstable blood supply and tumor metabolism, resulting in cycles of osmotic stress and recovery. Yet, how this osmotic stress - recovery cycle mediates tumorigenesis and cancer therapy remains poorly understood. Our unpublished results reveal sequential activation of molecular events during osmotic stress and recovery. In the stress stage, protein translation, transcription, and DNA replication are inhibited. Following recovery, protein translation resumes, gene transcription is greatly upregulated, but DNA replication is not immediately recovered. It turns out that a DNA replication checkpoint is activated during recovery; as a result, DNA replication is halted. Defects in activating protein translation inhibition during stress fail to activate the replication checkpoint during recovery, indicating a causative link from protein translation inhibition to DNA replication checkpoint activation. Many genes involved in this crosstalk are altered in a wide range of human tumors. Ovarian cancer cells that are resistant to standard of care (for instance, cisplatin) demonstrate defects in this crosstalk; however, they were also more sensitive to a combination of osmotic stress followed by the presence of replication checkpoint inhibitors during recovery than parental cancer cells. These findings highlight a unique role of the interstitial fluid system in tumor development and therapy response. They also suggest a new anticancer strategy by combining osmotic stress with checkpoint inhibitors. Key words: Tumor microenvironment; osmotic stress; protein translation control; DNA replication checkpoint; cancer therapy Citation Format: Fangfang Wang. Crosstalk between protein translation and DNA replication during stress response and cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3174.

Tolerance to replication stress requires Dun1p kinase and activation of the electron transport chain

One of the key outcomes of activation of DNA replication checkpoint (DRC) or DNA damage checkpoint (DDC) is the increased synthesis of the deoxyribonucleoside triphosphates (dNTPs), which is a prerequisite for normal progression through the S phase and for effective DNA repair.We have recently shown that DDC increases aerobic metabolism and activates the electron transport chain (ETC) to elevate ATP production and dNTP synthesis by repressing transcription of histone genes, leading to globally altered chromatin architecture and increased transcription of genes encoding enzymes of tricarboxylic acid (TCA) cycle and the ETC. The aim of this study was to determine whether DRC activates ETC. We show here that DRC activates ETC by a checkpoint kinase Dun1p-dependent mechanism. DRC induces transcription of RNR1-4 genes and elevates mtDNA copy number. Inactivation of RRM3 or SGS1, two DNA helicases important for DNA replication, activates DRC but does not render cells dependent on ETC. However, fitness of rrm3Δ and sgs1Δ cells requires Dun1p. The slow growth of rrm3Δdun1Δ and sgs1Δdun1Δ cells can be suppressed by introducing sml1Δ mutation, indicating that the slow growth is due to low levels of dNTPs. Interestingly, inactivation of ETC in dun1Δ cells results in a synthetic growth defect that can be suppressed by sml1Δ mutation, suggesting that ETC is important for dNTP synthesis in the absence of Dun1p function. Together, our results reveal an unexpected connection between ETC, replication stress, and Dun1p kinase.

DNA Replication Checkpoint Activates Respiration in Budding Yeast

In addition to DNA damage checkpoint (DDC), eukaryotic cells have also DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. We have showed previously that DDC activates respiration to increase ATP production and to elevate dNTP levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DNA damage‐induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain (ETC), and oxidative phosphorylation. Here we show that similarly to DDC, activation of DRC also induces respiration. However, unlike DDC, DRC does not affect histone gene expression and chromatin structure. DRC induces respiration by inducing transcription of RNR1‐4, upregulating synthesis of dNTPs, and elevating mtDNA copy number. Fitness of rrm3∆ and sgs1∆, mutants with defects in DNA replication, requires checkpoint kinase Dun1, but does not require ETC and oxidative metabolism. However, in the absence of Dun1, ETC is required for viability of rrm3Δ and sgs1Δ cells. In addition, inactivation of ETC in dun1Δ cells results in a synthetic growth defect. Together, our data show that when Dun1p function is compromised, rrm3Δand sgs1Δ cells depend on oxidative metabolism.

DNA Replication Checkpoint Activates Respiration in Budding Yeast

In addition to DNA damage checkpoint (DDC), eukaryotic cells have also DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. We have showed previously that DDC activates respiration to increase ATP production and to elevate dNTP levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DNA damage‐induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, and oxidative phosphorylation. Here we show that similarly to DDC, activation of DRC also induces respiration by a Mec1p‐ and Rad53p‐ dependent mechanism. However, unlike DDC, DRC induces respiration by a mechanism that is not dependent on histone gene expression and chromatin structure.Support or Funding InformationNIH GM120710

And-1 coordinates with Claspin for efficient Chk1 activation in response to replication stress.

The replisome is important for DNA replication checkpoint activation, but how specific components of the replisome coordinate with ATR to activate Chk1 in human cells remains largely unknown. Here, we demonstrate that And-1, a replisome component, acts together with ATR to activate Chk1. And-1 is phosphorylated at T826 by ATR following replication stress, and this phosphorylation is required for And-1 to accumulate at the damage sites, where And-1 promotes the interaction between Claspin and Chk1, thereby stimulating efficient Chk1 activation by ATR. Significantly, And-1 binds directly to ssDNA and facilitates the association of Claspin with ssDNA. Furthermore, And-1 associates with replication forks and is required for the recovery of stalled forks. These studies establish a novel ATR-And-1 axis as an important regulator for efficient Chk1 activation and reveal a novel mechanism of how the replisome regulates the replication checkpoint and genomic stability.

Maintain Genomic Stability: Multitask of DNA Replication Proteins.

Maintenance of genomic stability is critical for living organisms because it is crucial for cell survival and development, and it prevents the development of deleterious mutations. Overriding this control will cause genomic instability, a hallmark of cancer. The genome is highly vulnerable to damage, especially during DNA replication because chromosome is decondensed and the replication forks are extremely sensitive to DNA damage agents. The eukaryotic replisome, which consists of a large number of replication fork-associated proteins, is essential for the elongation of replication forks during DNA replication. This complex contains DNA polymerases, MCM helicase, single stranded DNA (ssDNA) binding protein RPA, sliding clamp PCNA, Tipin, Timeless, Claspin, And-1, etc. In cells with DNA damage such as replication stress, replication forks are stalled Figure 1. At stalled replication forks, some of replisome components switch their role from facilitating DNA synthesis to inducing activation of DNA replication checkpoint, a signaling transduction pathway that is critical to maintain fork stability and triggers cell cycle arrest.

Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB

Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB

Molecular Mechanisms of DNA Replication Checkpoint Activation

The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability.

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.

The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

Roles of Human AND-1 in Chromosome Transactions in S Phase

Coordinated execution of DNA replication, checkpoint activation, and postreplicative chromatid cohesion is intimately related to the replication fork machinery. Human AND-1/chromosome transmission fidelity 4 is localized adjacent to replication foci and is required for efficient DNA synthesis. In S phase, AND-1 is phosphorylated in response to replication arrest in a manner dependent on checkpoint kinase, ataxia telangiectasia-mutated, ataxia telangiectasia-mutated and Rad3-related protein, and Cdc7 kinase but not on Chk1. Depletion of AND-1 increases DNA damage, delays progression of S phase, leads to accumulation of late S and/or G2 phase cells, and induces cell death in cancer cells. It also elevated UV-radioresistant DNA synthesis and caused premature recovery of replication after hydroxyurea arrest, indicating that lack of AND-1 compromises checkpoint activation. This may be partly due to the decreased levels of Chk1 protein in AND-1-depleted cells. Furthermore, AND-1 interacts with cohesin proteins Smc1, Smc3, and Rad21/Scc1, consistent with proposed roles of yeast counterparts of AND-1 in sister chromatid cohesion. Depletion of AND-1 leads to significant inhibition of homologous recombination repair of an I-SceI-driven double strand break. Based on these data, we propose that AND-1 coordinates multiple cellular events in S phase and G2 phase, such as DNA replication, checkpoint activation, sister chromatid cohesion, and DNA damage repair, thus playing a pivotal role in maintenance of genome integrity.

An essential role for the RNA-binding protein Smaug during theDrosophilamaternal-to-zygotic transition

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.

Different requirements for the association of ATR–ATRIP and 9-1-1 to the stalled replication forks

DNA replication checkpoint, a surveillance mechanism for S-phase progression, plays a crucial role for the maintenance of genome integrity. A variety of factors have been characterized to be involved in the checkpoint signal transduction. Rpa, a single strand DNA binding protein, was found to be responsible for forming a structure that is recognized by checkpoint sensors and then emits the initial signal for the activation of DNA damage checkpoint. Here we use a mutant of rpa1 gene, rfa1-t11, that has defects in recruiting checkpoint sensor proteins to the site of double strand break, to examine the mutant's effects on the activation of DNA replication checkpoint. We found that the mutant cells activated DNA replication checkpoint normally and showed no defects in recruiting ATR–ATRIP, a major sensor complex that is essential for DNA replication/damage checkpoint, to the site of stalled forks. In contrast, the mutant was defective in recruiting 9-1-1 complex, another sensor complex that functions in DNA damage checkpoint signal transduction, to the stalled forks. Moreover we found that sensitivity for HU obviously appeared in rfa1-t11 mutant when Mrc1 was deleted, while deletion of Rad9, an adaptor specific for damage checkpoint, had subtle effect. These data strongly suggest that rfa1-t11 mutant was mainly defective for activating DNA damage checkpoint and molecular requirement for the recruitment of ATR–ATRIP and 9-1-1 to the stressed forks may be different.