Clastogenic Effect of the Human T-cell Leukemia Virus Type I Tax Oncoprotein Correlates with Unstabilized DNA Breaks

Franca Majone,Kuan-Teh Jeang

doi:10.1074/jbc.c000538200

Abstract

Expression of the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein rapidly engenders DNA damage as reflected in a significant increase of micronuclei (MN) in cells. To understand better this phenomenon, we have investigated the DNA content of MN induced by Tax. Using an approach that we termed FISHI, fluorescent in situ hybridization and incorporation, we attempted to characterize MN with centric or acentric DNA fragments for the presence or absence of free 3'-OH ends. Free 3'-OH ends were defined as those ends accessible to in situ addition of digoxigenin-dUTP using terminal deoxynucleotidyl transferase. MN were also assessed for centromeric sequences using standard fluorescent in situ hybridization (FISH). Combining these results, we determined that Tax oncoprotein increased the frequency of MN containing centric DNA with free 3'-OH and decreased the frequency of MN containing DNA fragments that had incorporation-inaccessible 3'-ends. Recently, it has been suggested that intracellular DNA breaks without detectable 3'-OH ends are stabilized by the protective addition of telomeric caps, while breaks with freely detectable 3'-OH are uncapped and are labile to degradation, incomplete replication, and loss during cell division. Accordingly, based on increased detection of free 3'-OH-containing DNA fragments, we concluded that HTLV-I Tax interferes with protective cellular mechanism(s) used normally for stabilizing DNA breaks.

Highlights

MN are small nuclei-like bodies found outside the main nucleus produced as consequences of chromosomal damage [7, 8]
One interpretation of these results is that this viral oncoprotein affects both the mismatch repair and the fidelity of chromosomal segregation functions postulated to be deficient in human cancers
Using in situ cytogenetic techniques (FISH and FISH and TdT end incorporation (FISHI)) and probes specific for centromeres or telomeres [13, 14], we have characterized the nature of corporation; DIG, digoxigenin; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine B isothiocyanate; DAPI, 4,6-diamidino2-phenylindole; Tdt, terminal deoxynucleotidyltransferase enzyme; Cen, centromeric); Te, telomeric; TI, TdT-accessible incorporation

Summary

Introduction

MN are small nuclei-like bodies found outside the main nucleus produced as consequences of chromosomal damage [7, 8] They exist at a low prevalence in cells ambiently and can be induced ex vivo by genotoxic compounds with different mechanisms of action. Yeast strains deficient for yKu and SIR proteins are hypersensitive to clastogenic agents [11, 12] One corollary of these observations is that termini of new breaks unprotected by telomeres with associated Ku, SIR, and/or other factors are labile. Such ends may not be efficiently repaired and may progress to larger lesions resulting in gross chromosomal aberrations which could provide the initiating basis for transformation [3]. Our finding that Tax disproportionately induces unprotected (free 3Ј-OH) DNA ends provides a molecular explanation for the clastogenic effect of this oncoprotein

Objectives

Methods

Results