Unstabilized DNA breaks in HTLV-1 Tax expressing cells correlate with functional targeting of Ku80, not PKcs, XRCC4, or H2AX

Cells mutated for PKcs, XRCC4 and H2AX showed increased frequency of MN and unstabilized DNA breaks in response to the expression of Tax, while cells genetically mutated for Ku80 were refractory to Tax’s induction of these cytogenetic effects. Moreover, by measuring the size of DIG-dUTP incorporation signal, which indicates the extent of unstable DNA ends, we found that Tax induces larger signals than those in control cells. However, in xrs-6 cells deficient for Ku80, this Tax effect was not seen.The data here demonstrate that clastogenic DNA damage in Tax expressing cells is explained by Tax targeting of Ku80, but not PKcs, XRCC4 or H2AX, which are all proteins directly or indirectly related to the non-homologous end-joining (NHEJ) repair system. Of note, the Ku80 protein plays an important role at the initial stage of the NHEJ repair system, protecting and stabilizing DNA-breaks. Accordingly, HTLV-1 Tax is shown to interfere with a normal cellular protective mechanism for stabilizing DNA breaks. These DNA breaks, unprotected by Ku80, are unstable and are subject to erosion or end-to-end fusion, ultimately leading to additional chromosomal aberrations.The HTLV-I Tax oncoprotein induces rapid cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) and unstabilized DNA breaks in cells [1-4]. Tax is thought to have both aneuploidogenic and clastogenic effects [3-7]. We previously characterized the phenomenon of Tax-associated clastogenic DNA damage by examining the status of DNA breaks in the nucleus and in the MN in the presence or absence of Tax [4-6]. In particular, we characterized DNA breaks for the presence or absence of free 3′-OH ends [4]. Free 3′-OH ends represent breaks accessible to the in situ addition of digoxigenin (DIG)-labeled dUTP using terminal deoxynucleotidyl transferase. On the other hand, an absence of accessible 3′-OH ends suggests that the breaks maybe protected by a protein complex(es).Unprotected f