Many studies have shown that folate deficiency induces DNA hypomethylation and DNA damage. DNA hypomethylation is thought to occur during DNA replication through deficiency of the DNA methylation donor synthesized using folate. However, the relationship between DNA hypomethylation and DNA damage caused by folate deficiency is not well understood. Here, we analyzed DNA damage caused by folate deficiency during the cell cycle S phase in which DNA hypomethylation occurs. Using HeLa cells, we first detected accumulation of intranuclear γ-H2A.X, a DNA double strand break marker, by immunofluorescent staining. We found that DNA damage occurs primarily during S phase, and that folate deficiency enhanced S phase-dependent DNA damage. We also found that folate deficiency caused slowing of the DNA replication fork and a delay in the onset of middle-late S phase. Detailed analyses of DNA damage in S phase progression suggested that the folate deficiency-induced DNA damage increased in all stages of S phase, particularly in middle-late S phase cells. To examine the relationship between DNA damage and DNA hypomethylation, we analyzed the methylation state of the promoter region of the human LINE1 repetitive sequence, where heterochromatin is formed and replicated during middle to late S phase. We found that DNA methylation decreased by about 10% under the folate deficiency condition. Taken together, our results suggest that DNA hypomethylation is associated with DNA damage caused by folate deficiency.
