DNA Ligase IV Prevents Replication Fork Stalling and Promotes Cellular Proliferation in Triple Negative Breast Cancer

Abstract

DNA damage is a hallmark of cancer, and mutation and misregulation of proteins that maintain genomic fidelity are associated with the development of multiple cancers. DNA double strand breaks are arguably considered the most deleterious type of DNA damage. The nonhomologous end-joining (NHEJ) pathway is one mechanism to repair DNA double strand breaks, and proteins involved in NHEJ may also regulate DNA replication. We previously established that DNA-PKcs, a NHEJ protein, promotes genomic stability and cell viability following cellular exposure to replication stress; we wanted to discern whether another NHEJ protein, DNA ligase IV (Lig4), shares this phenotype. Our investigations focused on triple negative breast cancer cells, as, compared to nonbasal breast cancer, LIG4 is frequently amplified, and an increased gene dose is associated with higher Lig4 expression. We depleted Lig4 using siRNA and confirmed our knockdown by qPCR and western blotting. Cell survival diminished with Lig4 depletion alone, and this was associated with increased replication fork stalling. Checkpoint protein Chk1 activation and dephosphorylation were unchanged in Lig4-depleted cells. Lig4 depletion resulted in sustained DNA-PKcs phosphorylation following hydroxyurea exposure. Understanding the effect of Lig4 on genomic replication and the replication stress response will clarify the biological ramifications of inhibiting Lig4 activity. In addition, Lig4 is an attractive clinical target for directing CRISPR/Cas9-mediated repair towards homology-directed repair and away from NHEJ, thus understanding of how diminishing Lig4 impacts cell biology is critical.

RNA:DNA hybrids from Okazaki fragments contribute to establish the Ku-mediated barrier to replication-fork degradation

“…Recent works support that NHEJ is active during DNA replication to repair stressed fork and to safeguard fork integrity 15 . In both yeast and mammals, several NHEJ-related factors, such as Ku, XLF, RIF1 and 53BP1, are engaged at stalled or broken fork to act as anti-resection barriers, independently of their canonical function in NHEJ [26][27][28][29][30][31][32][33] . Moreover, Ku binding at reversed or broken fork precedes HR activity, ensuring coordinated nascent strand degradation or Rad51 loading 27,34 .…”

Identification of LIG1 and LIG3 as prognostic biomarkers in breast cancer

“…The nonhomologous end-joining (NHEJ) pathway is a mechanism for repairing DNA double-strand break. DNA replication may be also regulated by the proteins involved in NHEJ [ 25 ]. In recent years, LIGs have been reported to be involved in the development of many cancers, such as lung cancer, nasopharyngeal cancer, rectal adenocarcinoma and BC [ 25 , 26 , 27 , 28 , 29 , 30 , 31 ].…”

“…DNA replication may be also regulated by the proteins involved in NHEJ [ 25 ]. In recent years, LIGs have been reported to be involved in the development of many cancers, such as lung cancer, nasopharyngeal cancer, rectal adenocarcinoma and BC [ 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. To our knowledge, the relationship between the expression of LIG1 and LIG3 and prognostic value for BC patients is still not clear.…”

Non-homologous end-joining at challenged replication forks: an RNA connection?

“…The cleaved fork is then repaired by the co-action of Rad52 and the Ligase4-Xrcc4 complex, known to join DNA ends during DSBs repair by NHEJ, allowing replication restart [13]. This type of EJ-dependent fork repair likely ensures, together with DNA-PKcs, proper cell proliferation and resistance to RS in human triple negative breast cancer [14].…”