Functional Analysis of the Replication Fork Proteome Identifies BET Proteins as PCNA Regulators

Wessel, Sarah R.; Mohni, Kareem N.; Luzwick, Jessica W.; Dungrawala, Huzefa; Chen, David

doi:10.1016/j.celrep.2019.08.051

Cited by 101 publications

(101 citation statements)

References 68 publications

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“…Decreased levels of these proteins in the proximity of replication forks may create an altered chromatin structure that negatively affects replication fork function or they may be directly involved in replisome function. Indeed, it was recently shown that Brd2, Brd3 and Brd4 function at the replication fork to antagonize the ATAD5-mediated unloading of PCNA, which is consistent with our observation that PCNA unloading is slowed in Hat1 -/cells (79). Finally, the presence of Hat1 on nascent chromatin near replication forks suggests that Hat1 may directly modify and regulate components of the replisome.…”

Section: Discussionsupporting

confidence: 92%

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

Garcia

Lovejoy

NAGARAJAN

et al. 2020

Preprint

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ABSTRACTThe replisome functions in a dynamic environment that is at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the action of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (Hat1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12 that accompanies replication-coupled chromatin assembly. Analysis of the role of Hat1 in replication-coupled chromatin assembly demonstrates that Hat1 also physically associates with chromatin near sites of DNA replication. The association of Hat1 with newly replicated DNA is transient but can be stabilized by replication fork stalling. The association of Hat1 with nascent chromatin may be functionally relevant as loss of Hat1 results in a decrease in replication fork progression and an increase in replication fork stalling. In addition, in the absence of Hat1, stalled replication forks are unstable and newly synthesized DNA becomes susceptible to Mre11-dependent degradation. These results suggest that Hat1 links replication fork function to the proper processing and assembly of newly synthesized histones.

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Section: Discussionsupporting

confidence: 92%

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

Garcia

Lovejoy

NAGARAJAN

et al. 2020

Preprint

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“…3A). After 72 hours of knock down, we wealth of studies that established mechanisms of BRD4 in transcription regulation, and earlier work showing replication dysfunction caused by BRD4 loss 30,38,67,68 , we focused on the role of BRD4 in the prevention of TRC-induced DNA damage.…”

Section: Bet Protein Lossmentioning

confidence: 99%

“…If these roadblocks are not resolved, collisions with the replication machinery will lead to replication fork breakdown and DNA strand breaks. Important factors have been identified that prevent and resolve R-loops, including the RNAPII activator CDK9 and the RNA:DNA hybrid endonuclease RNase H1 [27][28][29][30][31][32][33][34][35][36][37] .…”

Section: Introductionmentioning

confidence: 99%

BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation

Edwards

Maganti

Tanksley

et al. 2019

Preprint

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Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintain genomic integrity and cell survival. Deregulation of these systems can lead to conflicts between the transcription and replication machinery leading to DNA damage. BRD4, a BET bromodomain protein and known transcriptional regulator, interacts with P-TEFb to ensure efficient transcriptional elongation by stimulating phosphorylation of RNA Polymerase II (RNAPII). Here we report that disruption of BET bromodomain protein function causes DNA damage that correlates with RNAPII-dependent transcript elongation and occurs preferentially in S-phase cells.BET bromodomain inhibition also causes accumulation of RNA:DNA hybrids (R-loops), which are known to lead to transcription-replication conflicts, DNA damage, and cell death. Furthermore, we show that resolution of R-loops abrogates BET-bromodomain inhibitor-induced DNA damage, and that BET-bromodomain inhibition induces both Rloops and DNA damage at sites of BRD4 occupancy. Finally, we see that the BRD4 Cterminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET bromodomain inhibition. Together, these findings demonstrate that BET bromodomain inhibitors can damage DNA via induction of Rloops in highly replicative cells.

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“…In living cells many more proteins associate with replisomes than are required for “minimal” biochemical reconstructions 37,12 . These interactions may be constitutive or induced by replication stress, but are typically interpreted as representing a single complex (see Introduction) 15,22,38–42 .…”

Section: Discussionmentioning

confidence: 99%

“…The identification and characterization of the minimal components of biochemically active replisomes, the result of decades of extraordinary work from multiple laboratories, necessarily reflects studies with deproteinized model DNA substrates under carefully controlled conditions. However, in vivo there are hundreds of replisome associated proteins 8–12 . Presumably this reflects the multiple layers of complexity that characterize replication of the genome in living cells.…”

Section: Introductionmentioning

confidence: 99%

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Zhang

Bellani

James

et al. 2020

Preprint

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25Duplication of mammalian genomes requires replisomes to overcome numerous impediments 26 during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of 27 replication stress characterize mixed populations of challenged and unchallenged replication forks, 28 averaged across S phase, and model a single species of "stressed" replisome. However, in cells 29 containing potent obstacles to replication, we find two different lesion proximal replisomes. One 30 is bound by the DONSON protein and is more frequent in early S phase, in regions marked by 31 euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S 32 phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. CHIP-33 seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions 34 that replicate early and the skew of the latter towards regions that replicate late. 35Introduction 36 Eukaryotic replisomes are multiprotein complexes consisting, minimally, of the CMG 37 helicase [MCM2-7 (M), CDC45 (C), and GINS (go, ichi, ni, san) proteins (G)] which forms a ring 38 around the leading strand template. Other components include the pol α, ε, and δ polymerases, 39 MCM10, and a few accessory factors [1][2][3][4][5][6][7] . The identification and characterization of the minimal 40 components of biochemically active replisomes, the result of decades of extraordinary work from 41 multiple laboratories, necessarily reflects studies with deproteinized model DNA substrates under 42 carefully controlled conditions. However, in vivo there are hundreds of replisome associated 43 proteins 8-12 . Presumably this reflects the multiple layers of complexity that characterize replication 44 of the genome in living cells. For example, three dimensional analyses of chromosome structure 45 demonstrate two major domains. The A compartment contains euchromatin, which is accessible, 46 transcriptionally active, and marked by specific histone modifications, such as H3K4me3. The B 47 compartment, which is more condensed, contains inactive genes, many repeated elements, and is 48 associated with different histone modifications, including H3K9me3 13 . In addition to the structural 49 distinctions, regions of the genome are also subject to temporal control of replication during S 50 phase. Sequences in Compartment A tend to replicate early in S phase, while those in B are 51 duplicated in late S phase 14,15 . 52 Other influential effectors of replisome composition are the frequent encounters with 53 impediments, that stall or block either the progress of the CMG helicase or DNA synthesis. These 54 3 include alternate DNA structures, protein: DNA adducts, DNA covalent modifications introduced 55 by endogenous or endogenous reactants, depleted nucleotide precursor pools, etc. Replication 56 stress activates the ATR (ATM-and Rad3-related) kinase, with hundreds of substrates, including 57 MCM proteins [16][17][18] , and stimulates the recruitment of numerous facto...

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Functional Analysis of the Replication Fork Proteome Identifies BET Proteins as PCNA Regulators

Cited by 101 publications

References 68 publications

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

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