Abstract
Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea). In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats.
Highlights
Licensing of DNA replication origins begins early during the G1-phase of the cell cycle when ORC and CDC6 recruit CDT1-MCM2-7 to the chromatin [reviewed in 1]
Many red blood cell (RBC) micronuclei result from double strand DNA breaks that give rise to acentromeric chromosomal fragments that fail to incorporate into nuclei during mitosis and remain in the cell following enucleation
RBC micronuclear DNA is sequenced (Mic-Seq) to define the locations of breaks genome-wide and this assay is used to study ongoing genome instability resulting from insufficient DNA replication origin licensing
Summary
Animal husbandry programs and protocol reviews are in compliance with NIH, USDA, and New York State Standards. Mice were maintained in facilities covered under NIH assurance #A-3143-01, certified by New York State for the use of living animals, and the USDA APIHS registration as research facility #21–124. The studies were approved by the Roswell Park Cancer Institute Animal Care and Use Committee under Protocols 817M and 876M. Five to six week old wild type 129Sv and Mcm IRES-CreERT2/IRES-CreERT2 (MCM2 deficient) mice were used in studies addressing the effects of MCM2 deficiency. For studies addressing the effects of hydroxyurea (HU), 3 month old wild type 129Sv mice were administered HU continuously in the drinking water at the concentrations indicated in the text. Blood samples were taken by retro-orbital bleed or cardiac puncture
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