ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

Download Full-text

Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

Science Advances ◽

10.1126/sciadv.abc0330 ◽

2020 ◽

Vol 6 (38) ◽

pp. eabc0330 ◽

Cited By ~ 1

Author(s):

D. T. Gruszka ◽

S. Xie ◽

H. Kimura ◽

H. Yardimci

Keyword(s):

Dna Replication ◽

Real Time ◽

Single Molecule ◽

Replication Fork ◽

Epigenetic Inheritance ◽

Replication Forks ◽

Replication Fork Progression ◽

Egg Extracts ◽

Fork Stalling ◽

The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

Download Full-text

The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

Molecular and Cellular Biology ◽

10.1128/mcb.02190-06 ◽

2007 ◽

Vol 27 (8) ◽

pp. 3131-3142 ◽

Cited By ~ 151

Author(s):

Keziban Ünsal-Kaçmaz ◽

Paul D. Chastain ◽

Ping-Ping Qu ◽

Parviz Minoo ◽

Marila Cordeiro-Stone ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Protein A ◽

Chain Elongation ◽

Replication Forks ◽

Interacting Protein ◽

Checkpoint Response ◽

Replication Fork Progression ◽

Dna Chain ◽

Interfering Rna

ABSTRACT UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

Download Full-text

RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

Frontiers in Genetics ◽

10.3389/fgene.2021.753535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nathan Ellis ◽

Jianmei Zhu ◽

Mary K Yagle ◽

Wei-Chih Yang ◽

Jing Huang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Dna Helicase ◽

Replication Forks ◽

Replication Origins ◽

Dna Double Strand Breaks ◽

Response Factors ◽

Fork Stalling ◽

In Cells

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

Download Full-text

The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

10.1101/555003 ◽

2019 ◽

Author(s):

Magdalena Ganz ◽

Christopher Vogel ◽

Christina Czada ◽

Vera Jörke ◽

Rebecca Kleiner ◽

...

Keyword(s):

Cancer Cells ◽

Replication Fork ◽

Replication Stress ◽

Tumor Formation ◽

Replication Forks ◽

Functional Link ◽

Protein Levels ◽

Replication Fork Progression ◽

Dna Replication Stress ◽

Architectural Protein

ABSTRACTDNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

Download Full-text

Centrosome impairment causes DNA replication stress through MLK3/MK2 signaling and R-loop formation

10.1101/2020.01.09.898684 ◽

2020 ◽

Author(s):

Zainab Tayeh ◽

Kim Stegmann ◽

Antonia Kleeberg ◽

Mascha Friedrich ◽

Josephine Ann Mun Yee Choo ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Replication Stress ◽

List Type ◽

Replication Forks ◽

Loop Formation ◽

Animal Cells ◽

Replication Fork Progression ◽

Primordial Dwarfism ◽

Dna Replication Stress

AbstractCentrosomes function as organizing centers of microtubules and support accurate mitosis in many animal cells. However, it remains to be explored whether and how centrosomes also facilitate the progression through different phases of the cell cycle. Here we show that impairing the composition of centrosomes, by depletion of centrosomal components or by inhibition of polo-like kinase 4 (PLK4), reduces the progression of DNA replication forks. This occurs even when the cell cycle is arrested before damaging the centrosomes, thus excluding mitotic failure as the source of replication stress. Mechanistically, the kinase MLK3 associates with centrosomes. When centrosomes are disintegrated, MLK3 activates the kinases p38 and MK2/MAPKAPK2. Transcription-dependent RNA:DNA hybrids (R-loops) are then causing DNA replication stress. Fibroblasts from patients with microcephalic primordial dwarfism (Seckel syndrome) harbouring defective centrosomes showed replication stress and diminished proliferation, which were each alleviated by inhibition of MK2. Thus, centrosomes not only facilitate mitosis, but their integrity is also supportive in DNA replication.HighlightsCentrosome defects cause replication stress independent of mitosis.MLK3, p38 and MK2 (alias MAPKAPK2) are signalling between centrosome defects and DNA replication stress through R-loop formation.Patient-derived cells with defective centrosomes display replication stress, whereas inhibition of MK2 restores their DNA replication fork progression and proliferation.Graphical abstract

Download Full-text

Dual effect of heat shock on DNA replication and genome integrity

Molecular Biology of the Cell ◽

10.1091/mbc.e11-12-1009 ◽

2012 ◽

Vol 23 (17) ◽

pp. 3450-3460 ◽

Cited By ~ 36

Author(s):

Artem K. Velichko ◽

Nadezhda V. Petrova ◽

Omar L. Kantidze ◽

Sergey V. Razin

Keyword(s):

Dna Replication ◽

Heat Shock ◽

Replication Fork ◽

Stress Factors ◽

Dna Integrity ◽

Dependent Manner ◽

Replication Forks ◽

Replication Process ◽

Replication Fork Progression ◽

Dna Damage Signaling

Heat shock (HS) is one of the better-studied exogenous stress factors. However, little is known about its effects on DNA integrity and the DNA replication process. In this study, we show that in G1 and G2 cells, HS induces a countable number of double-stranded breaks (DSBs) in the DNA that are marked by γH2AX. In contrast, in S-phase cells, HS does not induce DSBs but instead causes an arrest or deceleration of the progression of the replication forks in a temperature-dependent manner. This response also provoked phosphorylation of H2AX, which appeared at the sites of replication. Moreover, the phosphorylation of H2AX at or close to the replication fork rescued the fork from total collapse. Collectively our data suggest that in an asynchronous cell culture, HS might affect DNA integrity both directly and via arrest of replication fork progression and that the phosphorylation of H2AX has a protective effect on the arrested replication forks in addition to its known DNA damage signaling function.

Download Full-text

Wss1 promotes replication stress tolerance by degrading histones

10.1101/2019.12.13.876110 ◽

2019 ◽

Author(s):

Karthik Maddi ◽

Daniel Kwesi Sam ◽

Florian Bonn ◽

Stefan Prgomet ◽

Eric Tulowetzke ◽

...

Keyword(s):

Dna Replication ◽

Protein Interactions ◽

Replication Fork ◽

Genome Stability ◽

Replication Stress ◽

Underlying Mechanism ◽

Adverse Conditions ◽

Replication Fork Progression ◽

Timely Completion ◽

Protein Crosslinks

SummaryTimely completion of DNA replication is central to accurate cell division and to the maintenance of genomic stability. However, certain DNA-protein interactions can physically impede DNA replication fork progression. Cells remove or bypass these physical impediments by different mechanisms to preserve DNA macromolecule integrity and genome stability. In Saccharomyces cerevisiae, Wss1, the DNA-protein crosslink repair protease, allows cells to tolerate hydroxyurea-induced replication stress but the underlying mechanism by which Wss1 promotes this function has remained unknown. Here we report that Wss1 provides cells tolerance to replication stress by directly degrading core histone subunits that non-specifically and non-covalently bind to single-stranded DNA. Unlike Wss1-dependent proteolysis of covalent DNA-protein crosslinks, proteolysis of histones does not require Cdc48 nor SUMO-binding activities. Wss1 thus acts as a multi-functional protease capable of targeting a broad range of covalent and non-covalent DNA-binding proteins to preserve genome stability during adverse conditions.

Download Full-text

SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

10.1101/2020.05.18.097154 ◽

2020 ◽

Author(s):

Julie Rageul ◽

Jennifer J. Park ◽

Ping Ping Zeng ◽

Eun-A Lee ◽

Jihyeon Yang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Essential Role ◽

Replication Stress ◽

Replication Forks ◽

Genomic Integrity ◽

Fork Protection Complex ◽

Stalled Replication Forks ◽

Stalled Fork

ABSTRACTProtecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

Download Full-text