Lawrence F. Povirk, Ph.D.
- University of California at Berkeley, 1977
DNA double-strand breaks are a highly toxic form of DNA damage, which are responsible for the killing of tumor cells by radiotherapy and some forms of chemotherapy. Low levels of DSBs produced by free radicals associated with oxidative metabolism are a major contributing factor to genomic instability and carcinogenesis. Our research is focused on the repair of DSBs, with particular emphasis on the resolution of damaged DNA ends prior to rejoining. Phosphoglycolate is a two-carbon sugar fragment formed at the 3′ ends of many free radical-mediated DSBs and PG removal is an obligatory early step in repair of such DSBs. We have identified two enzymes that are each capable of resolving 3′ PG ends, tyrosyl-DNA phosphodiesterase (TDP1) and Artemis.
TDP1 deficiency is associated with the very rare human genetic disease spinocerebellar ataxia with axonal neuropathy (SCAN1), whose symptoms include adolescent-onset ataxia (lack of coordination) and cerebellar atrophy. We have recently devised a novel method, based on ligation-mediated real-time PCR, to quantify 3′-PG termini on DSBs in intact human cells. We have thereby shown that PG termini are more persistent in SCAN1 than in normal lymphoblasts, confirming a role for TDP1 in repair of these lesions. In addition to PG termini, TDP1 also repairs covalent linkages between 3′ DNA ends and topoisomerase I (TopoI). TopoI relaxes supercoiling of DNA by introducing transient single-strand breaks and occasionally becomes irreversibly linked through tyrosine to the DNA 3′ end. To elucidate the relationships among TDP1 deficiency, SCAN1 and repair of 3′-PG and 3′-tyrosyl lesions, we have generated a TDP1-knockout mouse. Unfortunately, the TDP1-/- mouse does not recapitulate the behavioral pathology typical of SCAN1. Nevertheless, TDP1-/- cells derived from these mice show spontaneous chromosomal instability and sensitivity to agents that induce 3′-PG DSBs, indicating an important role for repair of this type of damage. However, because the TDP1 knockout mouse did not display typical symptoms of the human SCAN1 disease, we are also attempting to generate neurons lacking TDP1 from human stem cells, using the recently developed technique of somatic knockout by homologous recombination.
Artemis is involved in both the rejoining of DSBs formed by radiation and free radicals, and in the normal DNA breakage and rejoining by which antibody genes are assembled in lymphocytes. For this reason, Artemis deficiency in humans results in a specific form of severe combined immune deficiency that is accompanied by hypersensitivity to X-rays. Whereas TDP1 simply removes PG from a DNA end, Artemis, in conjunction with the DNA end-binding protein kinase DNA-PK, trims a very short (two to four bases) segment of DNA from the DNA end. We have constructed cells which have a mutant form of Artemis that lacks the trimming activity, and these cells are as radiosensitive as those that have no Artemis protein at all. Moreover, they show the same DSB repair deficiency, suggesting that it is the trimming activity that is essential for repair of radiation damage. Recent data suggest that Artemis is specifically required for repair of DSBs in highly condensed heterochromatin, and thus we are attempting to adapt our ligation-mediated real-time PCR assay to determine whether PG ends at DSBs in heterochromatic DNA are more persistent when Artemis is absent.
A third project investigates the role of another DSB repair factor known as XLF, which is involved in the replacement of DNA bases that are destroyed by free radicals when the DSB is formed. We are using an iterative ribosomal peptide synthesis and selection procedure to identify small peptides that will bind to XLF at its interface with another repair protein, XRCC4, and inhibit formation of the DSB repair complex. These molecules may be useful for blocking DSB repair and thus improving the effectiveness of radiation therapy and some forms of chemotherapy.
Zhou RZ, Akopiants K and Povirk LF. (2010) Patching and single-strand ligation in nonhomologous DNA end joining despite persistence of a closely opposed 3′-phosphoglycolate-terminated strand break. Radiation Research. 174(3):274-9.
Adams BR, Hawkins AJ, Povirk LF and Valerie K. (2010) ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells. Aging (Albany, N.Y.). 2(9):582-96.
Akopiants K, Zhou RZ, Mohapatra S, Valerie K, Lees-Miller SP, Lee KJ, Chen DJ, Revy P, de Villartay JP and Povirk LF. (2009) Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts. Nucleic Acids Research. 37(12):4055-62.
Hawkins AJ, Subler MA, Akopiants K, Wiley JL, Taylor SM, Rice AC, Windle JJ, Valerie K and Povirk LF. (2009) In-vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation. DNA Repair (Amst). 8(5):654-63.
Yannone SM, Khan IS, Zhou RZ, Zhou T, Valerie K and Povirk LF. (2008) Coordinate 5′ and 3′ endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase. Nucleic Acids Research. 36(10):3354-65.
Povirk LF, Zhou T, Zhou R, Cowan MJ and Yannone SM. (2007) Processing of 3′-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease. The Journal of Biological Chemistry. 282(6):3547-58.