Mapping Structurally Defined Guanine Oxidation Products along DNA Duplexes: Influence of Local Sequence Context and Endogenous Cytosine Methylation

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S-phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin which extends up to one kilobase from early, efficient replication origins. The CMG holo-helicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex, but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

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Generation, repair and replication of guanine oxidation products

Genes and Environment ◽

10.1186/s41021-017-0081-0 ◽

2017 ◽

Vol 39 (1) ◽

Cited By ~ 34

Author(s):

Katsuhito Kino ◽

Masayo Hirao-Suzuki ◽

Masayuki Morikawa ◽

Akane Sakaga ◽

Hiroshi Miyazawa

Keyword(s):

Oxidation Products ◽

Guanine Oxidation

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Spectroscopic Study of Cytosine Methylation Effect on Thermodynamics of DNA Duplex Containing CpG Motif

Journal of Spectroscopy ◽

10.1155/2015/842810 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Václav Římal ◽

Ondřej Socha ◽

Josef Štěpánek ◽

Helena Štěpánková

Keyword(s):

Cytosine Methylation ◽

Accurate Determination ◽

Joint Analysis ◽

Dna Duplexes ◽

Temperature Dependences ◽

Scattering Spectra ◽

Enthalpy And Entropy ◽

Cpg Motif ◽

Raman Scattering Spectra ◽

Duplex Formation

Effect of cytosine methylation on DNA duplexes was studied by using a model system of three self-complementary DNA octamers containing central CpG motif surrounded by a couple of AT base pairs, CAACGTTG, CATCGATG, and CTTCGAAG, and their analogues with the central cytosine methylated at C5 position. Temperature dependences of1H NMR, UV absorption, and Raman scattering spectra measured for aqueous solutions at concentrations of different orders of magnitude were subjected to a joint analysis that allowed an accurate determination of the enthalpy and entropy of duplex formation. It was revealed that the changes of the enthalpy and entropy contributions are strongly dependent on the base composition in the vicinity of the CpG motif.

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Isolation, Characterization, and Independent Synthesis of Guanine Oxidation Products

European Journal of Organic Chemistry ◽

10.1002/ejoc.200500581 ◽

2006 ◽

Vol 2006 (6) ◽

pp. 1351-1378 ◽

Cited By ~ 56

Author(s):

Thanasis Gimisis ◽

Crina Cismaş

Keyword(s):

Oxidation Products ◽

Guanine Oxidation ◽

Independent Synthesis

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Carcinogen-induced DNA structural distortion differences in the RAS gene isoforms; the importance of local sequence

BMC Chemistry ◽

10.1186/s13065-021-00777-8 ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Georgina E. Menzies ◽

Ian A. Prior ◽

Andrea Brancale ◽

Simon H. Reed ◽

Paul D. Lewis

Keyword(s):

Context Effects ◽

Ras Gene ◽

Sequence Context ◽

Methyl Group ◽

Lung Cancer Patients ◽

Local Distortion ◽

Mutational Pattern ◽

Local Sequence ◽

Modelling Techniques ◽

Gene Isoforms

Abstract Background Local sequence context is known to have an impact on the mutational pattern seen in cancer. The RAS genes and a smoking carcinogen, Benzo[a]pyrene diol epoxide (BPDE), have been utilised to explore these context effects. BPDE is known to form an adduct at the guanines in a number of RAS gene sites, KRAS codons 12, 13 and 14, NRAS codon 12, and HRAS codons 12 and 14. Results Molecular modelling techniques, along with multivariate analysis, have been utilised to determine the sequence influenced differences between BPDE-adducted RAS gene sequences as well as the local distortion caused by the adducts. Conclusions We conclude that G:C > T:A mutations at KRAS codon 12 in the tumours of lung cancer patients (who smoke), proposed to be predominantly caused by BPDE, are due to the effect of the interaction methyl group at the C5 position of the thymine base in the KRAS sequence with the BPDE carcinogen investigated causing increased distortion. We further suggest methylated cytosine would have a similar effect, showing the importance of methylation in cancer development.

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Locally acting transcription factors are required for p53-dependent cis-regulatory element activity

10.1101/761387 ◽

2019 ◽

Author(s):

Allison N. Catizone ◽

Gizem Karsli Uzunbas ◽

Petra Celadova ◽

Sylvia Kuang ◽

Daniel Bose ◽

...

Keyword(s):

Transcription Factors ◽

Regulatory Element ◽

Regulatory Elements ◽

Mrna Levels ◽

Sequence Context ◽

Endogenous Expression ◽

Damage Repair ◽

Local Sequence ◽

Element Activity ◽

Massively Parallel Reporter Assay

AbstractThe master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CRE), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors (TFs). Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. We identified a SP1/KLF family motif located in an intronic p53 CRE that is required for the endogenous expression of the p53-dependent gene CCNG1. We also identified ATF3 as a factor that co-regulates the expression of the p53-dependent gene GDF15 through binding with p53 in an upstream CRE. Loss of either p53 or ATF3 severely reduces CRE activity and alters endogenous GDF15 mRNA levels in the cell. Our data suggests that p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that p53 activity is guarded against loss of any one regulatory partner allowing for dynamic and redundant control of p53-mediated transcription.

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