Petra Pfeiffer

Chromosomal aberrations: formation, identification and distribution

Chromosomal aberrations (CA) are the microscopically visible part of a wide spectrum of DNA changes generated by different repair mechanisms of DNA double strand breaks (DSB). The method of fluorescence in situ hybridisation (FISH) has uncovered unexpected complexities of CA and this will lead to changes in our thinking about the origin of CA. The inter- and intrachromosomal distribution of breakpoints is generally not random. CA breakpoints occur preferentially in active chromatin. Deviations from expected interchromosomal distributions of breakpoints may result from the arrangement of chromosomes in the interphase nucleus and/or from different sensitivities of chromosomes with respect to the formation of CA. Telomeres and interstitial telomere repeat like sequences play an important role in the formation of CA. Subtelomeric regions are hot spots for the formation of symmetrical exchanges between homologous chromatids and cryptic aberrations in these regions are associated with human congenital abnormalities.

Long-term XPC silencing reduces DNA double-strand break repair

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPC(KD) cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC(KD) cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPC(KD) cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC(KD) and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.

Use of dna end joining activity of a xenopus laevis egg extract for construction of deletions and expression vectors for hiv-1 tat and rev proteins

We have developed a cloning strategy which combines conventional T4 DNA ligation with the highly efficient nonhomologous DNA end joining (EJ) activity of an extract from Xenopus laevis eggs. The nonhomologous EJ activity allowed the rapid construction of deletion mutants by the intramolecular rejoining of nonhomologous DNA ends generated for the purpose of deleting restriction fragments from the vector. The combined use of T4 DNA ligase for intermolecular ligation and X. laevis egg extracts for intramolecular nonhomologous EJ proved to be a powerful tool, as demonstrated here for the construction of expression vectors for HIV-1 Tat and Rev.

Activation of a system for the joining of nonhomologous DNA ends during Xenopus egg maturation.

Mature Xenopus laevis eggs provide an elementary reaction system of illegitimate recombination which efficiently joins nonhomologous DNA ends (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). Here we show that stage VI oocytes, known to express a system for homologous recombination (D. Carroll, Proc. Natl. Acad. Sci. USA 80:6902-6906, 1983), are completely devoid of this joining system. Nonhomologous DNA end-to-end joining, however, attains full activity only at an extremely late stage of egg maturation. Cycloheximide inhibition patterns indicate that nonhomologous joining activity is regulated at the G2 restriction point of the cell cycle. Implications of homologous and nonhomologous recombination activities during egg maturation are discussed.

Analysis of double-strand break repair by nonhomologous DNA end joining in cell-free extracts from mammalian cells.

Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.

Radiosensitivity and double-strand break rejoining in tumorigenic and non-tumorigenic human epithelial cell lines

Radiosensitivity and repair of DNA damage induced by ionizing radiation and restriction enzymes were investigated in three human epithelial cell lines: two tumorigenic squamous carcinoma cell lines (SCC-4 and SCC-25), and a non-tumorigenic epidermal keratinocyte cell line (RHEK-1). Sensitivity to ionizing radiation was determined using a clonogenic cell survival assay, which showed SCC-4 to be more radiosensitive than SCC-25 and RHEK-1, which in turn displayed about equal sensitivity. Using DNA precipitation under alkaline conditions for the analysis of induction and repair of DNA single-strand breaks (ssb), an increased level of ssb induction was found for SCC-4 while the efficiency of ssb repair was about equal in all three cell lines. Using pulsed-field gel electrophoresis (PFGE) for the measurement of induction and repair of DNA double-strand breaks (dsb), no consistent differences were detected between the three cell lines. A plasmid reconstitution assay was used to determine the capacity to rejoin restriction enzyme-induced dsb in whole-cell extracts prepared from the three cell lines. In these experiments, dsb rejoining was shown to be significantly reduced in the most radiosensitive SCC-4 cell line while it was about equal in RHEK-1 and SCC-25. The results indicate that plasmid reconstitution in cell-free extracts is a sufficiently sensitive assay to detect differences in repair capacity among tumour cell lines of different radiosensitivity which remain undetectable by DNA precipitation and PFGE.

Expression of the TrkA or TrkB receptor tyrosine kinase alters the double-strand break (DSB) repair capacity of SY5Y neuroblastoma cells.

In the childhood tumor neuroblastoma, high expression of the TrkA neurotrophin receptor is associated with a favorable prognosis and a lack of structural chromosomal changes, whereas TrkB is expressed in aggressive neuroblastomas demonstrating high genomic instability. The ability to repair DNA double-strand breaks (DSBs) is considered a central determinant of chromosomal stability with nonhomologous end joining (NHEJ) being the major pathway of DSB repair in vertebrates. Here, we used the SH-SY5Y human neuroblastoma cell line ectopically expressing either TrkA or TrkB as a model system to analyze the impact of Trk receptor expression on NHEJ-mediated DSB repair. In a cell-free NHEJ assay, SY5Y-TrkA cells displayed a significantly higher efficiency for NHEJ compared to SY5Y-TrkB cells. To detect possible underlying mechanisms, gene expression data (Affymetrix U95A microarray chips) obtained from the same SY5Y-TrkA/TrkB model system were reanalyzed focussing on genes involved in DNA repair. Expression of XRCC4, a central component of NHEJ, was significantly upregulated in SY5Y-TrkA compared to SY5Y-TrkB cells. Expression data were confirmed using real-time PCR and western blotting. Additionally, XRCC4 expression was enhanced in most primary neuroblastomas with high TrkA expression. The TrkA-induced increase in NHEJ activity could be reverted by XRCC4 knock-down, confirming the induction of XRCC4 by TrkA to be essential for the observed phenotype. Our data provide the first evidence for a functional relationship between tyrosine kinase receptor signaling and NHEJ-mediated DSB repair in cancer cells, potentially contributing to their genomic stability.

A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA

Background: Double‐strand breaks in chromosomal DNA of eucaryotic cells are assumed to be repaired by mechanisms of illegitimate recombination capable of direct rejoining of the broken ends. Cell‐free extracts of Xenopus laevis eggs efficiently perform these end joining reactions with any pair of noncomplementary DNA termini whose single‐stranded 5′‐ or 3′‐overhangs do not exceed a length of ≈ 10 nt.

Mechanisms of Non-Homologous DNA End Joining:Aspects of In Vitro Assays

Double-strand breaks (DSB) in genomic DNA are a major threat to cell survival and chromosome integrity. In vertebrate cells, non-homologous DNA end joining (NHEJ) is the major pathway of DSB repair. Genetic studies in yeast, human and rodent cell lines displaying increased IR sensitivity and defects in DSB repair have provided insight in the genes involved in NHEJ. These genetic data have been confirmed and complemented by in vitro assays which have played a significant role in the elucidation and the dissection of the basic mechanisms underlying NHEJ. In vitro assays utilize model DNA substrates that carry defined DSB and thus provide information on the efficiency and fidelity of NHEJ in different cell systems. In contrast to investigations in living cells, in vitro assays facilitate the investigation of the functions of single proteins in the repair process itself so that their impact on the rejoining of different DNA end structures can be studied directly without interference by other cellular processes such as cell cycle and replication. In this chapter, we summarize the basic features of in vitro assays and give an overview over the different available cell-free systems which have facilitated the detailed analysis of NHEJ mechanisms in different vertebrate cells.

DNA-Reparatur und Mutagenese

Die Weitergabe genetischer Information ist ein auserst akkurater, aber nie ganz fehlerfrei ablaufender Vorgang. Er erlaubt die Ausstattung der Tochterzellen mit dem bewahrten, fast identischen Genom der Mutterzelle. Die Mutationsrate, d.h. die Veranderung der genetischen Information im Zeitraum einer Zellteilungsphase, ist abhangig vom Organismus, Zelltyp und dem betrachteten Allel. Daher ist die Fahigkeit, Mutationsraten kontrollieren zu konnen, eine herausragende Leistung jeder einzelnen Zelle. Erreicht wird dies durch einen hinreichend genauen Mechanismus der Replikation und das Vorhandensein von Reparatursystemen, die auftretende DNA-Schaden rechtzeitig eliminieren.