DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells

DNA Repair ◽  
2012 ◽  
Vol 11 (12) ◽  
pp. 976-985 ◽  
Author(s):  
Daniel Vare ◽  
Petra Groth ◽  
Rickard Carlsson ◽  
Fredrik Johansson ◽  
Klaus Erixon ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks ◽  
Replication Block

DNA Interstrand Crosslinks Repair in Mammalian Cells

2008 ◽  
Vol 63 (3-4) ◽  
pp. 289-296 ◽  
Author(s):  
Veronika Mladenova ◽  
George Russev
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Competent Cell ◽  
Gfp Expression ◽  
Considerable Part ◽  
Strand Breaks ◽  
Interstrand Crosslinks ◽  
Dna Interstrand Crosslinks ◽  
Native Plasmid

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by γ-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of γ-H2AX foci was observed when cells were transfected with native plasmid, which indicated that γ-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HRproficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates

1987 ◽  
Vol 7 (1) ◽  
pp. 129-140
Author(s):  
F L Lin ◽  
K M Sperle ◽  
N L Sternberg
Keyword(s):  
Mammalian Cells ◽  
Essential Feature ◽  
Double Strand Breaks ◽  
The Other ◽  
Strand Breaks ◽  
Double Stranded Dna ◽  
Linear Molecules ◽  
Recombinant Molecule ◽  
Dna Substrates ◽  
Extrachromosomal Recombination

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'----3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'----5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tk delta 5' and tk delta 3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'----3' degradative model and the other would be the favored substrate in the alternative 3'----5' degradative model. Our results indicate that the intermediate favored by the 3'----5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

scholarly journals Involvement of DNA polymerase μ in the repair of a specific subset of DNA double-strand breaks in mammalian cells

2007 ◽  
Vol 35 (11) ◽  
pp. 3551-3560 ◽  
Author(s):  
Jean-Pascal Capp ◽  
François Boudsocq ◽  
Anne-Gaelle Besnard ◽  
Bernard S. Lopez ◽  
Christophe Cazaux ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Specific Subset

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

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