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.