Free radical-mediated DNA double strand breaks (DSBs) are induced either directly by ionizing radiation or by certain chemicals like bleomycin. These breaks are terminated by 3'-PG (PO4CH2COOˉ) or 3'-phosphate groups formed as a result of fragmentation of deoxyribose. To study the nature of repair of these 3'-blocked breaks, we constructed substrates mimicking free-radical induced DSBs. Human and yeast tyrosyl DNA-phosphodiesterase (Tdpl) efficiently processed substrates with 3'-PGs, in either the presence or absence of magnesium, to give a 3'-phosphate. Gel filtration chromatography and western blotting codmed that the putative enzyme in human extracts that efficiently processed PG was indeed tyrosyl DNA-phosphodiesterase. When recombinant hTdpl was purified using HiTrap nickel chelating columns and its PG processing activity compared to that of partially purified native enzyme (from lymphoblastoid whole-cell extracts using Sephacryl S-300 gel filtration columns), we found that the recombinant enzyme had lesser 3'-PG removal activity than the partially purified native enzyme. On cloning recombinant FLAG-tagged hTdpl into human expression vectors, we observed that the FLAG epitope tag did not show any evidence of affecting the specificity of the enzyme. Due to the many differences between bacterial and human cells, we cloned recombinant FLAG-tagged hTdpl into U-87 cells (adenovirus infected glioma cell) and this recombinant enzyme showed the same specificity toward PG substrates as when prepared from bacteria. End-processing assays using the NHEJ proteins- Ku, DNA-PK and XRCC4/Ligase IV-alone or in combination showed an inhibition of hTdpl activity on 3'- overhangs. In nuclear extracts, hTdp1 association with XRCC1, a single-strand repair protein, showed to increase the PG-processing activity of Tdpl up to 4 times. Whole-cell extracts containing mutant Tdpl derived from patients suffering from spinocerebellar axonal neuropathy (SCAN1) were found to be deficient in PG-processing. Addition of JRLl whole-cell extract (SCAN1 extract containing mutant Tdpl) to purified FLAG-tagged hTdpl showed to decrease the phosphotyrosyl processing and increase the PG-processing of FLAG-tagged hTdpl suggesting that there must be other factors in the extract that affect the enzyme activity. Experiments carried out to check for the presence of Tdpl in mitochondrial extracts obtained from GM1310 normal human fibroblasts as well as in SCANl (JRL) mitochondrial extracts, showed that mitochondrial extracts contained Tdpl at a concentration comparable to whole-cell extracts. Our results also showed that mitochondrial extracts from the SCANl cell-line, JRL3 (containing mutant Tdpl), lacked detectable Tdpl activity suggesting that all PG-processing activity in mitochondria may be attributable to Tdpl.