Project description:G-quadruplexes (G4s) form throughout the genome and influence important cellular processes, but their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected, dual role for the dsDNA translocase HLTF in G4 metabolism. First, we find that HLTF is enriched at G4s in the human genome and suppresses G4 accumulation throughout the cell cycle using its ATPase activity. This function of HLTF affects telomere maintenance by restricting alternative lengthening of telomeres, a process stimulated by G4s. We also show that HLTF and MSH2, a mismatch repair factor that binds G4s, act in independent pathways to suppress G4s and to promote resistance to G4 stabilization. In a second, distinct role, HLTF restrains DNA synthesis upon G4 stabilization by suppressing PrimPol-dependent repriming. Together, the dual functions of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
Project description:Telomere dysfunctional CMP/GMP have deregulated pathways that are associated with DNA damage signaling We compared differentially expressed genes in the G4/G5 CMP relative to the G0 control to identify pathways that may affect CMP differentiation. Bone marrow CMP and GMP cells were sorted from two paired pools of G0 TERTER/+ or G4/G5 TERTER/ER mice (5,000-20,000 cells per sample) using the Influx Cell Sorter. Every paired pool includes CMP or GMP sorted from 4 age and gender matched G0 or G4/G5 mice. RNA from the respective sorted cells was extracted using Trizol (Ambion) and profiled on 2100 Bioanalyzer (Agilent). Gene expression profiling was performed at the Sequencing and Non-coding RNA Program at MD Anderson Cancer Center. Briefly, the GeneChip® 3 IVT Express Kit (Affymetrix) was used to generate biotin-labeled cRNA, which were purified and fragmented, before target hybridization on the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix) according to the manufacturer's instructions. Affymetrix raw data (CEL files) were normalized using Affymetrix Microarray Suite (MAS) version 5.0 using a TGT=100. Paired pools used in the study were: CMP pool 1: G0-1 and G4/5-1 CMP pool 2: G0-2 and G4/G5-2 GMP pool 1: G0-1 and G4/G5-1 GMP pool 2: G0-2 and G4/G5-2
