Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks

Abstract

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

“…Further, we confirmed the differential activity of HR and NHEJ pathways in mESC and fibroblasts by in vitro functional assays [39][40][41]. Recombination reactions using nuclear protein extracts prepared from exponentially growing cells showed a 3-fold increase in HR activity in mESC compared to primary fibroblasts (P value <0.001; Fig.…”

“…The NHEJ in vitro assay was conducted as described by Liang et al [40,41]. Briefly, the linearized DNA substrate was incubated with nuclear protein extract in 50 mM Tris-HCL (pH 7.6), 10 mM MgCl 2 , 1 mM dithiothreitol, 1 mM ATP, and 25% polyethylene glycol 8000 at 148C for 2 h. Aftr the reaction, DNA was deproteinized and purified by using a PCR purification kit (Qiagen).…”

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos

“…In yeast and human cells, LIG4 mediates double-strand ligation during NHEJ [46,47], and Lig3 is considered a necessary factor for homologous end ligation [7,21,22]. We next determined which ligases are required for MMEJ activity in zebrafish embryos.…”

“…By contrast, NHEJ is the most straightforward way to repair a DSB, as the broken ends are simply rejoined regardless of the genetic sequences at the break. Efficient NHEJ is most active during G1 and early S phase, and it requires Ku70/Ku80 heterodimers, DNA-PK catalytic subunits in mammalian cells have shown that PARP-1, ligase 3, CtIP, and Mre11 can promote MMEJ [7,11,[21][22][23][24]. Nevertheless, how often MMEJ occurs during vertebrate development and which type of ligase is required for MMEJ repair in vivo remain largely unknown.…”

Lack of Association Between LIG4 Gene Polymorphisms and the Risk of Breast Cancer: A HuGE Review and Meta-analysis

“…Therefore, variants in genes involved in DNA DSB repair are considered tobe good candidates for breast cancer susceptibility. LIG4 encoding the protein DNA Ligase IV, is a human ATP-dependent DNA ligase TMR, the rate of total mutation; CMR, the rate of complete mutation; PMR, the rate of partial mutation; OR, odds ratio; 95%CI, 95% confidence interval gene that plays a significant role in joining double-strand breaks during the NHEJ pathway of double-strand break repair (Liang et al, 2008 (Dapic et al, 2005). However, Kuschel et al indicated that Ligase IV can decrease the risk for breast cancer.…”