Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

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Stratification of pediatric ALL by in vitro cellular responses to DNA double-strand breaks provides insight into the molecular mechanisms underlying clinical response

Blood ◽

10.1182/blood-2008-03-142950 ◽

2009 ◽

Vol 113 (1) ◽

pp. 117-126 ◽

Cited By ~ 13

Author(s):

Eliot Marston ◽

Victoria Weston ◽

Jennifer Jesson ◽

Esther Maina ◽

Carmel McConville ◽

...

Keyword(s):

Molecular Mechanisms ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Cellular Responses ◽

Double Strand Breaks ◽

Induction Treatment ◽

Dna Double Strand Breaks ◽

Primary Resistance ◽

Strand Breaks

Abstract The molecular basis of different outcomes in pediatric acute lymphoblastic leukemia (ALL) remains poorly understood. We addressed the clinical significance and mechanisms behind in vitro cellular responses to ionizing radiation (IR)–induced DNA double-strand breaks in 74 pediatric patients with ALL. We found an apoptosis-resistant response in 36% of patients characterized by failure to cleave caspase-3, -7, -9, and PARP1 by 24 hours after IR and an apoptosis-sensitive response with the cleavage of the same substrates in the remaining 64% of leukemias. Resistance to IR in vitro was associated with poor early blast clearance at day 7 or 15 and persistent minimal residual disease (MRD) at day 28 of induction treatment. Global gene expression profiling revealed abnormal up-regulation of multiple prosurvival pathways in response to IR in apoptosis-resistant leukemias and differential posttranscriptional activation of the PI3-Akt pathway was observed in representative resistant cases. Importantly, pharmacologic inhibition of selected prosurvival pathways sensitized apoptosis-resistant ALL cells to IR in vitro. We suggest that abnormal prosurvival responses to DNA damage provide one of the mechanisms of primary resistance in ALL, and that they should be considered as therapeutic targets in children with aggressive disease.

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MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks

Cell ◽

10.1016/j.cell.2006.03.013 ◽

2006 ◽

Vol 124 (6) ◽

pp. 1299 ◽

Cited By ~ 1

Author(s):

Manuel Stucki ◽

Julie A. Clapperton ◽

Duaa Mohammad ◽

Michael B. Yaffe ◽

Stephen J. Smerdon ◽

...

Keyword(s):

Cellular Responses ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Histone H2ax

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Change in DNA after Exposure to Hydrogen Atoms.

Zeitschrift für Naturforschung B ◽

10.1515/znb-1969-1216 ◽

1969 ◽

Vol 24 (12) ◽

pp. 1565-1573 ◽

Cited By ~ 8

Author(s):

H. Jung, ◽

U. Hagen, ◽

M. Ullrich, ◽

E. E. Petersen

Keyword(s):

Hydrogen Bonds ◽

Double Strand Breaks ◽

Single Strand ◽

Dna Double Strand Breaks ◽

Hydrogen Atoms ◽

Γ Irradiation ◽

Base Damage ◽

Strand Breaks ◽

Single Strand Breaks ◽

Single Breaks

The action of hydrogen atoms — generated in an electrodeless high frequency gas discharge — on calf thymus DNA in aqueous solution was investigated. The loss of priming activity was compared with the appearance of single strand breaks in native and denatured DNA, double strand breaks, denatured zones, base damage and rupture of hydrogen bonds. The primary lesions after exposure to H atoms and gamma radiation, respectively, are single strand breaks and base damage. Double strand breaks originating from accumulation of single breaks, and rupture of hydrogen bonds caused by single breaks and base damage, were identified as secondary lesions. In relation to strand breaks arising from radical attack on the sugar-phosphate backbone of the DNA molecule, base damage is about 12.5 times more frequent after Η-exposure than after γ-irradiation. It is concluded from this observation, that single strand breaks are the predominant critical lesions responsible for the loss of the functional activity of DNA.

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