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Lawrence F. Povirk,
Ph.D. Professor 401 College Street Massey Cancer Center Goodwin Research Laboratories Room 380A Box 980035 Richmond, Virginia 23298-0035 Phone: (804) 828-9640 E-mail: lpovirk@vcu.edu Publications: selected | PubMed |
Education: University of California at Berkeley, 1977
Research interests:
DNA double-strand breaks (DSBs) are a highly toxic form
of DNA damage, which are responsible for 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 (PG)
is a 2-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 (SSBs), and occasionally
becomes irreversibly linked through a 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 (SCID) 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 (2-4 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 absence.
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. Radiat Res. 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 NY). 2010 Sep;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 Res. 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, aand 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 Res. 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. J Biol Chem. 282(6):3547-58.