Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells

Abstract

Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70 ؊/؊ cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.Symmetrical exchanges between newly replicated chromatids and their sisters can be visualized cytologically in vertebrate cells if the DNA of one chromatid is labelled with 5-bromodeoxyuridine (BUdR) during synthesis. Sister chromatid exchanges (SCEs) can be induced by various genotoxic treatments (10), suggesting that SCEs reflect a DNA repair process. Cytological assessment of SCE levels in peripheral blood lymphocytes is used as an index of the mutagenic potential of environmental factors. More importantly, ϳ10 SCEs occur spontaneously in normally cycling human cells (5, 8), suggesting a link between SCE and DNA replication. Elevated spontaneous SCE levels are observed in cells from Bloom syndrome patients (9), in mouse cells that lack poly(ADP-ribose) polymerase (29) or KU70 (15), and in hamster cells with defects in XRCC1 (28), but the causal relationships between these enzymes and SCE are not clear.While the phenomenon of SCE has long been established (27) and many observations about the induction of SCEs have been made, their molecular basis remains obscure. SCE is intimately associated with DNA replication, and eukaryotic cells exposed to DNA-damaging agents in G 2 show elevated SCE levels only after completing a subsequent replication cycle (32). Homologous recombination (HR) was suggested as one of the mechanisms responsible (13,14). While HR occurs between sister chromatids in yeast as a means to replicate around UV-induced lesions (12), it has not been considered constitutively active during metazoan mitosis, perhaps because of the predominance of the nonhomologous DNA end-joining (NHEJ) pathway (30). In addition, the lack of recombinational repair mutants precluded direct testing of HR's involvement in SCE, so other models evolved. It was proposed that SCEs result from strand switching at stalled replication forks (20). Another model involved topoisomerase II action at coincident breaks at replication for...

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

“…24 SCEs can be monitored by the incorporation of bromodeoxyuridine (BrdU) for two DNA duplication cycles, which leads to a staining gap in one chromatid and a gain of staining in the corresponding sister chromatid (Fig. 3C).…”

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

“…The major cellular consequence of a BLM defect is an increase in homologous recombination (SCE and homologous chromatid interchanges) and in the rate of widespread mutations (German, 1993). It has been recently con®rmed that homologous recombination is the mechanism responsible for SCE, indicating that DNA lesions accompanying replication can be repaired by homologous recombination using the nascent sister chromatid as template (Sonoda et al, 1999). Thus, when an unrepaired lesion blocks replication fork progression, two alternative mechanisms can take place: the lesion can be by-passed by translesion synthesis or repaired by homologous recombination through sister chromatid exchanges.…”

Sister chromatid exchange in selected horse breeds (<i>Equus caballus</i>)

“…Sister chromatid exchanges are induced by many factors that disturb DNA structure, metabolism and repair mechanisms. The frequency of SCE increases as a result of DNA damage by factors that inhibit the progression of the replication fork in replicons (Sonoda et al 1999). Faulty systems of DNA damage repair (e.g.…”

“…Faulty systems of DNA damage repair (e.g. homologic recombination) also cause SCEs (Sonoda et al 1999, Wilson & Thompson 2007.…”