Complex genetic interactions between DNA polymerase β and the NHEJ ligase

Abstract

Mammalian cells possess multiple pathways for repairing various types of DNA damage. Although the molecular mechanisms of each DNA repair pathway have been analyzed by biochemical analysis and cell biological analysis, interplay between different pathways has not been fully elucidated. In this study, using human Nalm‐6–mutant cell lines, we analyzed the relationship between the base excision repair factor DNA polymerase β (POLβ) and DNA ligase IV (LIG4), which is essential for DNA double‐strand break (DSB) repair by non‐homologous end‐joining (NHEJ). We found that cells lacking both POLβ and LIG4 grew significantly more slowly than either single mutant, indicating cooperative functions of the two proteins in normal cell growth. To further investigate the genetic interaction between POLβ and LIG4, we examined DNA damage sensitivity of the mutant cell lines. Our results suggested that NHEJ acts as a backup pathway for repairing alkylation damage (when converted into DSBs) in the absence of POLβ. Surprisingly, despite the critical role of POLβ in alkylation damage repair, cells lacking POLβ exhibited increased resistance to camptothecin (a topoisomerase I inhibitor that induces DNA single‐strand breaks), irrespective of the presence or absence of LIG4. A LIG4‐independent increased resistance associated with POLβ loss was also observed with ionizing radiation; however, cells lacking both POLβ and LIG4 were more radiosensitive than either single mutant. Taken together, our findings provide novel insight into the complex interplay between different DNA repair pathways.

A Cell System-Assisted Strategy for Evaluating the Natural Antioxidant-Induced Double-Stranded DNA Break (DSB) Style

“…The alkylating agent, MMS, alkylates guanine and adenine to 7-methylguanine and 3-methyladenine, respectively [26]. Further, the alkylated base is repaired via base excision repair (BER), and in our previous study, we reported that accumulated base damage not repaired by BER is converted to DSBs in BER-proficient Nalm-6 cells and that at least some DSBs are repaired by NHEJ [18,21]. One-ended DSBs also occur in MMS-treated human A549 cells [27].…”

“…The human pre-B cell line, Nalm-6, and its derivative were cultured in a 5% CO 2 incubator at 37 • C in minimum essential medium (MEM; Nissui Seiyaku, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (GE Healthcare, Marlborough, MA, USA), 2 mM MEM non-essential amino acids (Wako Pure Chemical, Osaka, Japan), 1 mM sodium pyruvate (Wako Pure Chemical), 50 µM 2-mercaptoethanol (Wako Pure Chemical), and 0.15 mM vitamin B12 (Sigma-Aldrich, St. Louis, MO, USA) [21]. The human cervical cancer cell line, HeLa, was obtained from the RIKEN Bioresource Research Center and cultured in a 5%-CO 2 incubator at 37 • C in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (GE Healthcare) and 100 U/mL of penicillin/streptomycin (Wako Pure Chemical).…”

“…The growth inhibition assay was performed as described previously [21]. Briefly, 2 × 10 4 cells were seeded into 24-well plates and cultured for 96 or 192 h in a growth medium containing various concentrations of bleomycin (Wako Pure Chemical), etoposide (BioVision, Mountain View, CA, USA), cisplatin (Sigma-Aldrich), camptothecin (Sigma-Aldrich), sulforaphane (Cayman Chemical Company, Ann Arbor, MI, USA), resveratrol (Wako Pure Chemical), quercetin (Sigma-Aldrich), kaempferol (Sigma-Aldrich), or genistein (Wako Pure Chemical).…”

“…The colony formation assay was performed as described previously [21]. Briefly, 1 × 10 2 -10 5 cells were seeded into 60 cm dishes containing 5 mL of agarose medium with various concentrations of methyl methanesulfonate (MMS; Sigma-Aldrich).…”

Biological Adaptations of Tumor Cells to Radiation Therapy

“…However, many details are still unknown ( 69 ). For example, leukemia cells lacking DNA pol β cannot perform the BER pathway efficiently but can activate the NHEJ pathway to repair damage by alkylation ( 70 ).…”