Wolfgang Goedecke

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.

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.

Double Strand Break Repair Mechanisms in Mammalian Cells

Double-strand breaks (DSBs) represent major threats in chromosomal DNA. They arise either as intermediate structures during recombination, replication and repair events or as potentially lethal lesions introduced by ionising radiation or drugs. In any case, DSBs have to be eliminated immediately, because of the recombinogenic capacity of the DNA ends generated by the DSB that increases the risk for undesired chromosomal aberrations (CAs). In order to cope with DSBs, cells exhibit two different sets of repair activities, namely non-homlogous end joining (NHEJ) and homologous recombinational repair (HRR). Both pathways are regulated during the cell cycle with peak activities either in G1 phase (NHEJ) or in late S and G2 phases (HRR). While NHEJ has the capacity to join arbitrary DNA ends together, HRR depends on the presence of a second undamaged sequence in the genome providing a template for the reconstitution of the sequence at the DSB site. While most of the proteins identified so far participate in one of the two repair pathways, a few proteins are known to participate in both pathways. These last group of proteins is supposed to be involved in the selection process of the DNA repair pathway. Mutations in one DSB repair system result in the accumulation of DSBs and increased levels of CAs. As a result, genome instability is observed in cells with impaired DSB repair functions.

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.

Mutagenese und DNA-Reparaturmechanismen

Die Weitergabe der genetischen Information ist ein auserst akkurater, aber nie ganz fehlerfrei ablaufender Vorgang. Gewahrleistet wird diese hohe Genauigkeit durch die chemische Stabilitat des DNA-Molekuls, einen hinreichend genauen Replikationsmechanismus und das Vorhandensein von Reparatursystemen, die aufgetretene DNA-Schaden eliminieren. Kommt es trotzdem zu einer vererbbaren Veranderung der genetischen Information, wird von einer Mutation gesprochen.