Replication independent DNA double-strand break retention may prevent genomic instability.
BACKGROUND
Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, gamma-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability.


RESULTS
We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the gamma-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of gamma-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51.


CONCLUSIONS
Methylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.
Kongruttanachok
Narisorn
N
Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
Phuangphairoj
Chutipa
C
Thongnak
Araya
A
Ponyeam
Wanpen
W
Rattanatanyong
Prakasit
P
Pornthanakasem
Wichai
W
Mutirangura
Apiwat
A
D000107:Acetylation; D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D019175:DNA Methylation / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D042822:Genomic Instability / Q000235:genetics*; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors / Q000633:toxicity; D006657:Histones / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000633:toxicity; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D025521:Tumor Suppressor Proteins; C012589:trichostatin A; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases