Ku Protein Levels, Localization and Association to Replication Origins in Different Stages of Breast Tumor Progression

Abstract

Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis.

The Ku heterodimer: Function in DNA repair and beyond

“…The expression of Ku has been frequently found deregulated in tumor samples and its expression level has been proposed as a marker of predicting patient response to radiation therapy and survival [213][214][215][216][217][218]. The exploitation of genetic defects in DNA repair and DDR factors have long been employed in cancer treatment [219].…”

The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer

“…For example, up-regulation of Ku70 and Ku80 protein levels was found to correlate with tumor proliferation rate in non-melanoma skin cancer [ 161 ]. Increased expression and chromatin bound levels of Ku80 are also implicated as a driver of breast cancer progression [ 162 ]. In rectal carcinomas, increased Ku expression correlates with decreased disease-free survival [ 163 ].…”

Proximity Labeling Techniques to Study Chromatin

“…Another example of coupling proximity and affinity purification with mass spectrometry is a study investigating the interactome of the DNA repair factor Ku70 (Abbasi and Schild-Poulter, 2019). Besides its well-known role in nonhomologous end-joining, Ku70 is also implicated in other chromatin processes, e.g., transcriptional regulation or DNA replication (Mo and Dynan, 2002;Abdelbaqi et al, 2013). BioID identified a total of 501 candidates across three biological replicates, while AP-MS detected 282.…”