Resonance assignment and secondary structure of the tandem harmonin homology domains of human RTEL1.

Regulator of telomere elongation helicase 1 (RTEL1) is an Fe-S cluster containing DNA helicase that plays important roles in telomere DNA maintenance, DNA repair, and genomic stability. It is a modular protein comprising an N-terminal helicase domain, two tandem harmonin homology domains 1 & 2 (HHD1 and HHD2), and a C-terminal C4C4 type RING domain. The N-terminal helicase domain disassembles the telomere t/D-loop and unwinds the G-quadruplex via its helicase activity. The C-terminal RING domain interacts with telomere DNA binding protein TRF2 and helps RTEL1 recruitment to the telomere. The tandem HHD1 and HHD2 are characterized as a putative protein-protein interaction domain and have recently been shown to interact with a DNA repair protein SLX4. Several mutations associated with Hoyeraal-Hreidarsson syndrome and pulmonary fibrosis have been found in HHD1 and HHD2 of RTEL1. However, these domains have not been characterized for their structures. We have expressed and purified HHD1 and HHD2 of human RTEL1 for their characterization using solution NMR spectroscopy. Here, we report near complete backbone and sidechain 1H, 13C and 15N chemical shift assignments and secondary structure of the HHD1 and HHD2 domains of human RTEL1.

DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in Saccharomyces cerevisiae.

Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS.