scholarly journals The intersection of DNA replication with antisense 3′ RNA processing in Arabidopsis FLC chromatin silencing

2021 ◽  
Vol 118 (28) ◽  
pp. e2107483118
Author(s):  
Colette L. Baxter ◽  
Saša Šviković ◽  
Julian E. Sale ◽  
Caroline Dean ◽  
Silvia Costa
Keyword(s):  
Single Molecule ◽  
Antisense Rna ◽  
Replication Fork ◽  
Replication Forks ◽  
Line System ◽  
Replication Fork Progression ◽  
Fiber Analysis ◽  
Floral Repressor ◽  
Processing Factors ◽  
Chicken Dt40 Cell

How noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3′ processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3′ processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC.

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

2020 ◽  
Vol 6 (38) ◽  
pp. eabc0330 ◽  
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.

scholarly journals Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

2018 ◽  
Author(s):  
Mathew Stracy ◽  
Adam J.M. Wollman ◽  
Elzbieta Kaja ◽  
Jacek Gapinski ◽  
Ji-Eun Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Replication Fork ◽  
Dna Gyrase ◽  
Chromosomal Dna ◽  
Replication Fork Progression ◽  
Dna Strands ◽  
Steady State Levels ◽  
Negative Supercoiling ◽  
Supercoil Relaxation

ABSTRACTBacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in liveEscherichia colicells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

scholarly journals Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro Cicconi ◽  
Rekha Rai ◽  
Xuexue Xiong ◽  
Cayla Broton ◽  
Amer Al-Hiyasat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Clinical Feature ◽  
Replication Forks ◽  
Primary Microcephaly ◽  
Dna Damage And Repair ◽  
Homology Directed Repair ◽  
Replication Fork Progression ◽  
Telomere Binding Protein ◽  
Telomere Replication

AbstractTelomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1–TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

scholarly journals XLF and H2AX function in series to promote replication fork stability

2019 ◽  
Vol 218 (7) ◽  
pp. 2113-2123 ◽  
Author(s):  
Bo-Ruei Chen ◽  
Annabel Quinet ◽  
Andrea K. Byrum ◽  
Jessica Jackson ◽  
Matteo Berti ◽  
...  
Keyword(s):  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
End Joining ◽  
Replication Fork Progression ◽  
In Series ◽  
Non Homologous End Joining ◽  
C Complex ◽  
Factor C ◽  
Atr Kinase

XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7–dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax−/−:Xlf−/− cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
Author(s):  
Jessica A. Vaisica ◽  
Anastasija Baryshnikova ◽  
Michael Costanzo ◽  
Charles Boone ◽  
Grant W. Brown
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
E3 Ubiquitin Ligase ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Binding Partners ◽  
Efficient Recovery ◽  
Stalled Replication Forks

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.

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

2007 ◽  
Vol 27 (8) ◽  
pp. 3131-3142 ◽  
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.

scholarly journals Chk1 Requirement for High Global Rates of Replication Fork Progression during Normal Vertebrate S Phase

2006 ◽  
Vol 26 (8) ◽  
pp. 3319-3326 ◽  
Author(s):  
Eva Petermann ◽  
Apolinar Maya-Mendoza ◽  
George Zachos ◽  
David A. F. Gillespie ◽  
Dean A. Jackson ◽  
...  
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Fork Progression ◽  
Chk1 Inhibitor ◽  
Interfering Rna ◽  
First Time ◽  
Dt40 Cells

ABSTRACT Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1 −/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1 −/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3 −/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1 −/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1 −/− cells are associated with the accumulation of aberrant replication fork structures.

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