scholarly journals Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts

1999 ◽  
Vol 19 (3) ◽  
pp. 2206-2211 ◽  
Author(s):  
Agnes M. Cordonnier ◽  
Alan R. Lehmann ◽  
Robert P. P. Fuchs
Keyword(s):  
Xeroderma Pigmentosum ◽  
Replication Fork ◽  
Molecular Mechanisms ◽  
Translesion Synthesis ◽  
Distinct Pattern ◽  
Skin Fibroblasts ◽  
Primer Extension ◽  
Normal Cells ◽  
Replication Fork Progression ◽  
Dna Strands

ABSTRACT Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3′ end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.

scholarly journals Mechanistic Model of Replication Fork Progression in Yeast

2021 ◽  
Author(s):  
Ghanendra Singh
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
Replication Fork ◽  
Essential Role ◽  
Molecular Mechanisms ◽  
Molecular Level ◽  
Mechanistic Model ◽  
Normal Cells ◽  
Replication Fork Progression ◽  
Mammalian System

Replication fork progression complex plays an essential role during DNA replication. It travels along with the DNA with a particular speed called replication fork speed. Faithful duplication of the genome requires strict control over replication fork speed. Both acceleration and pausing mechanisms of the replication fork complex are regulated at the molecular level. Based on the experimental evidence, DNA replicates faster in normal cells than cancer cells, whereas cancer cells duplicate themselves more quickly than normal cells. Then in principle, accelerating the replication fork complex in cancer cells beyond a specific threshold speed limit can cause DNA damage and plausibly kill them. A modular mathematical model is proposed to explain the dynamics of replication fork control during DNA replication using the underlying molecular mechanisms in yeast which can extend to the mammalian system in the future.

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 Replication fork dynamics and the DNA damage response

2012 ◽  
Vol 443 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Rebecca M. Jones ◽  
Eva Petermann
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Replication Fork ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Replication Initiation ◽  
Developmental Defects ◽  
Replication Fork Progression ◽  
Damage Response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

scholarly journals A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rebecca A. Dagg ◽  
Gijs Zonderland ◽  
Emilia Puig Lombardi ◽  
Giacomo G. Rossetti ◽  
Florian J. Groelly ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
High Throughput Sequencing ◽  
Germline Mutations ◽  
Cdk Inhibitor ◽  
Rna Seq ◽  
Gene Encoding ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Transcriptional Alterations

AbstractBRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

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