RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase

RTEL1 contributes to DNA replication and repair and telomere maintenance

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embr

Flow cytometry analysis and sorting of chromosomes following hybridization with fluorescent probes that target specific DNA repeat sequences

Traditional cytogenetic approaches allow analysis of the chromosomal composition (karyotype) of mitotic cells fixed on slides cells by microscopy. The combination of karyotyping and Fluorescence In Situ Hybridization (FISH) enables the detection of specific target sequences on individual chromosomes. Disadvantages are that traditional cytogenetic approaches are very labor and time consuming and that chromosome specific information from only a few dozen cells has poor statistical power. An alternative is flow karyotyping, a method to analyze chromosomes in suspension by flow cytometry. For flow karyotyping, the DNA composition of specific chromosomes in suspension is measured based on the DNA-specific dyes Hoechst 33258 (HO) and Chromomycin A3 (CA3). My thesis work has focused on the development of a new method to analyze and sort chromosomes using FISH with labeled peptide nucleic acid (PNA) probes on chromosomes in suspension. I found that, following FISH, flow karyotyping can be used to detect and quantify repetitive DNA sequences within individual chromosomes. Using chromosome flow FISH (CFF), chromosomes isolated from cells of various species were hybridized to PNA probes and analyzed by flow cytometry. CFF was used to detect a variety of repeats; interstitial telomeric sequences in Chinese Hamster chromosomes, major satellite in mouse chromosomes and D18Z1 alpha satellite repeats in human chromosomes. Quantitative measurements of repeat length by CFF were validated by comparison with measurements obtained using Q-FISH. We found that parental homologs of human chromosome 18 with different D18Z1 satellite repeat array size could be purified using CFF and Fluorescence Activated Cell Sorting (FACS). Illumina short read sequencing of libraries built from these purified chromosomes enabled us to determine, with a high resolution, the allelic phasing of each homolog over the entire chromosome 18. Finally, CFF was modified to study sister chromatids separately. Using a cell model with inducible separation of sister chromatids, flow karyograms were generated. Using chromosome orientation FISH (CO-FISH) in suspension, we could identify sister chromatids according to the presence of DNA template strands. We anticipate that this approach will allow the purification of sister chromatids to study epigenetic differences between sister chromatids defined on the basis of DNA template strands.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Epigenetic differences between sister chromatids?

Semi-conservative replication ensures that the DNA sequence of sister chromatids is identical except for replication errors and variation in the length of telomere repeats resulting from replicative losses and variable end processing. What happens with the various epigenetic marks during DNA replication is less clear. Many chromatin marks are likely to be copied onto both sister chromatids in conjunction with DNA replication, whereas others could be distributed randomly between sister chromatids. Epigenetic differences between sister chromatids could also emerge in a more predictable manner, for example, following processes that are associated with lagging strand DNA replication. The resulting epigenetic differences between sister chromatids could result in different gene expression patterns in daughter cells. This possibility has been difficult to test because techniques to distinguish between parental sister chromatids require analysis of single cells and are not obvious. Here, we briefly review the topic of sister chromatid epigenetics and discuss how the identification of sister chromatids in cells could change the way we think about asymmetric cell divisions and stochastic variation in gene expression between cells in general and paired daughter cells in particular

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA