Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Fernández, José Luís; Vázquez-Gundı́n, Fernando; Rivero, María Teresa; Goyanes, Vicente; Gosálvez, Jaime

doi:10.1016/s0027-5107(00)00146-9

Cited by 32 publications

(18 citation statements)

References 25 publications

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“…This agrees with previous findings reporting that the major satellite DNA in mouse, localized in pericentromeric areas, is the chromosome domain contributing most actively to the production of single stranded DNA. Additionally, an identical situation was reported by Fernández et al (2001) in the pericentromeric 5-bp classical satellite DNA sequence as well as in the constitutive heterochromatic Yqh region of human somatic cells, and by López-Fernández et al (2006) in the grasshopper Pyrgomorpha conica. In the latter species, in fact, an abundance of ALS was observed in the penta poly-AATGG DNA, the telomere DNA sequence for this species, which in this case, as in the Chinese hamster CHO9 cell line (Mosquera et al, 2005), forms extremely large blocks in pericentromeric constitutive heterochromatin.…”

Section: Discussionsupporting

confidence: 70%

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Dávila-Rodríguez

Cortés‐Gutiérrez

López‐Fernández

et al. 2009

Cytogenet Genome Res

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Whole-comparative genomic hybridization (W-CGH) allows identification of chromosomal polymorphisms related to highly repetitive DNA sequences localized in constitutive heterochromatin. Such polymorphisms are detected establishing competition between genomic DNAs in an in situ hybridization environment without subtraction of highly repetitive DNA sequences, when comparing two species from closely related taxa (same species, sub-species, or breeds) or somewhat related taxa. This experimental approach was applied to investigating differences in highly repetitive sequences of three sheep breeds (Castellana, Ojalada, and Assaf). To this end, W-CGH was carried out using mouflon (sheep ancestor) chromosomes as a common target to co-hybridize equimolar quantities of two genomic DNAs obtained from either Castellana, Ojalada or Assaf sheep breeds. The results showed that the amount of constitutive heterochromatin is greater in all pericentromeric heterochromatin regions of acrocentric chromosomes than in metacentric or sex chromosomes. Additionally, when W-CGH was performed using DNAs from the Iberian breeds Castellana and Ojalada, chromosomal pericentromeric regions revealed quantitatively and qualitatively a presence of DNA families similar to that obtained from any of the above-cited breeds. On the contrary, when the DNA used in W-CGH experiments was obtained from Assaf, as compared to either Castellana or Ojalada, two different pericentromeric DNA families of highly repetitive sequences could be detected. Lastly, sex chromosomes were shown to be homogeneous among all breeds and thus revealed no detectable constitutive heterochromatin. W-CGH results were confirmed using DNA breakage detection-FISH experiments (DBD-FISH) carried out on lymphocytes. As a whole, the results showed that two different repetitive DNA families are present in the pericentromeric heterochromatin of the sheep breeds studied here. Additionally, they suggest a differential presence of these distinct repetitive DNA families in Castellana and Ojalada breeds as compared to the Assaf breed. Finally, the results of W-CGH after using mouflon as the targeted chromosomes also show that the two DNA families are present in the ancestor.

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Section: Discussionsupporting

confidence: 70%

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Dávila-Rodríguez

Cortés‐Gutiérrez

López‐Fernández

et al. 2009

Cytogenet Genome Res

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“…It is noteworthy that when a whole-genome DNA probe is hybridized to somatic cells, the background DBD-FISH signal is not homogeneous and certain chromatin regions are selectively and strongly labeled; this is especially evident when high levels of alkali denaturation are used on the native sperm chromatin (Figure 3, DBD-FISH High). It is of interest to highlight that the DNA sequences related with constitutive ALS mostly correspond with certain specific highly repetitive DNA sequences (Fernández et al, 2001; Rivero et al, 2001, 2004). In human leukocytes, the more intense background DBD-FISH areas within the genome correspond to DNA domains containing 5-bp satellite DNA sequences (Fernández and Gosálvez, 2002).…”

Section: Sperm Dna Comet Under Alkaline Denaturing Conditionsmentioning

confidence: 99%

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

et al. 2014

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Key Concepts The two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar–phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites “ALSs.”DBD–FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility.

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“…Our ChIP-seq data mapped many of the sites of uS3 binding to DNA to pericentromeric domains and nucleolus-associated chromatin, which is enriched in the centromeric and pericentromeric DNA (26,44). These chromosomal domains contain a large fraction of alpha satellite repeats, which are prone to form loops with a significant exposure of ssDNA (45–47). Moreover, the amount of AP sites in alpha satellite DNA is greatly increased in comparison with regular DNA (45).…”

Section: Discussionmentioning

confidence: 99%

“…These chromosomal domains contain a large fraction of alpha satellite repeats, which are prone to form loops with a significant exposure of ssDNA (45–47). Moreover, the amount of AP sites in alpha satellite DNA is greatly increased in comparison with regular DNA (45). When we mapped the repetitive reads on the human genome, the frequency of alpha satellites was about 30% higher than in the control without the antibodies, which indicates that uS3 could also be associated with alpha satellite DNA.…”

Section: Discussionmentioning

confidence: 99%

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit

Grosheva

Zharkov

Stahl

et al. 2017

Nucleic Acids Research

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Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked mainly to the 5΄-part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolus-associated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.

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Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Cited by 32 publications

References 25 publications

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit

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