Abstract
Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3-4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t1/2 on = 2.0 min) and was exceptionally stable, with an off rate of t1/2= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.
Highlights
Mouse mammary tumor virus is a slow transforming retrovirus that causes mammary tumors in lactating mice [1, 2]
Nuclear Factor Binding to the Separated Upper and Lower Strands of the MMTV LTR—We have previously demonstrated in electrophoretic mobility shift assays (EMSA) and in single-stranded DNA footprinting experiments with the thymidine specific reagent KMnO4, that nuclear factors in human Jurkat T Cell nuclear extracts bound to both the upper and lower single strands of the MMTV LTR over NRE1 [20]
To increase our understanding of the interaction of nuclear factors with the separated single strands of the MMTV LTR in and around NRE1, we examined nuclear factor binding by single-stranded DNase I protection footprinting (Fig. 1)
Summary
Mouse mammary tumor virus is a slow transforming retrovirus that causes mammary tumors in lactating mice [1, 2]. Has been demonstrated that the region of the viral LTR between Ϫ420 and Ϫ360 contains sequences that act to repress viral transcription in several cell types, but most notably in T cells [13,14,15,16,17,18,19,20] These sequences appear to be important for restricting cellular transformation by MMTV to the mammary gland, as viruses containing deletions encompassing this region of the LTR induce T cell lymphoma in addition to mammary carcinoma [19, 21,22,23,24,25,26]. We demonstrated that this sequence functioned as a direct, sequence-specific DNA binding site for Ku autoantigen/ DNA-dependent protein kinase (DNA-PK) [27] Both Ku and the DNA-PK catalytic subunit (DNA-PKcs) were found to be required for the transcriptional effects of NRE1 on MMTV expression [27]
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