(provided by candidate): Environmental and endogenous oxidants and toxicants can damage genomic DNA. Base excision repair (BER) is responsible for repairing such cytotoxic and mutagenic lesions that if not corrected can lead to deleterious mutations, genomic instability, or cell death. The multi-step BER pathway is coordinated by hand-off or channeling of DNA repair intermediates between the gap filling DNA synthesis step by DNA polymerase ? (pol ?) and ligation step by DNA ligase. However, pol ? exhibits structural adjustments upon correct versus incorrect or oxidized nucleotide insertion into the gap and this impacts substrate channeling to the ligation step. The molecular mechanism of the hand-off to the ligation step of the BER pathway remains unclear. My preliminary data suggest that DNA ligase fails and abortive ligation occurs after pol ? insertion of an incorrect or oxidized nucleotide. Furthermore, the modified structure of the resulting DNA intermediate after pol ? mismatch extension coupled to gap filling leads to failed ligation. The goal of the proposed work is to examine whether ligation failure in the last step of BER as an important source of genomic instability and cytotoxicity in mammalian cells.
In Aim 1, I will examine the effects of correct an incorrect nucleotide insertion during gap filling DNA synthesis coupled to ligation. For this purpose, I will measure nucleotide insertion kinetics in the presence of DNA ligase using wild-type pol ? and active site mutants and then compare the rates and extents of ligation. I will use various types of DNA substrates to address the effects of insertion of oxidized or incorrect nucleotide.
In Aim 2, I will evaluate the effects of other BER proteins, 3'-trimming enzymes, ligation conditions, and ligase forms for correcting or modifying failed ligation. I will evaluate other BER and 3'-end processing proteins in correcting impaired coordination during gap filling coupled ligation. Ligase reaction conditions and other forms of DNA ligase protein with impaired pol ? interaction also will be examined for their roles in modifying failed ligation.
In Aim 3, I ill determine the effects of DNA ligase deficiency on cellular cytotoxicity after oxidant and toxicant exposure. The possible link between cell phenotype and pol ?-mediated oxidized base insertion with accumulation of toxic BER intermediates will be examined using in vivo cell survival assays. BER and ligation failure with environmental agent-induced cytotoxic lesions also will be quantified by measuring the amount of abortive ligation product in BER intermediates. Completion of these aims will increase the understanding of biochemical and cytotoxic effects of premature or failed DNA ligation during BER compromised by oxidant and environmental toxicant-induced effects.
Cytotoxic and mutagenic lesions arise from many environmental agents, and they promote deleterious DNA damage leading to genomic instability and cell death. This negative cellular impact is prevented by DNA repair, but in the case of repair failure toxic consequences occur. To better understand these harmful effects, this grant application will examine how the structural adjustments of a DNA polymerase impact downstream steps of the DNA repair pathway that protects cells from the effects of endogenous stressors and environmental toxicants.
| Çaglayan, Melike; Wilson, Samuel H (2017) Role of DNA polymerase ? oxidized nucleotide insertion in DNA ligation failure. J Radiat Res 58:603-607 |
