Direct observation of thymine dimer repair in DNA by photolyase Ya Ting Kao Chai

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Direct observation of thymine dimer repair in DNA by photolyase Ya Ting Kao Chaitanya Saxena Lijuan Wang', Aziz Sancar and Dongping Zhong" of Physics, chemistry, and Biochemistry, Programs of Biophysio Physici, and Biochemistry, Ohio State University, 124 West 1Bth Avenue, Columbus, OH 43210, and University Carolina School Mary Ellen ohns Building C 72s0. Chapel Ha, NC This contribution is part of the special series of Inaugural Articles by membersofthe National Academy of Sdenceselected on May 2005. Contributed by Aziz sankar, August 2.200s Photolyase uses light energy to split uv-induced cydobutane E coli photolyase was prepared as in ref 15. The dimers in damaged DNA, but its molecular mechanism has never photoantenna molecule methenyltetrahydrofol ate was removed been directly revealed. Here, we report the direct mapping of during purification by photodecomposition (60. For all femto- catalytic processes through femtosecond synchronization of the concentration of 400 pM was enzymatic dynamics with the repair function. We observed direct used in reaction buffer, containing 50 mMTris (pH 741. 50 mM electron transfer from the excited flavin cofactor to the dimer in mM EDTA, 10 maM DTT and 50s (vol/vol glycerol. 170ps and back electron transfer from the repaired thymines in S60 The flavin cofactor in all purified samples contains the neutral ps. Both reactions are strongly modulated by active-site solvation radical FAD form. To reduce the flavin cofantor to the to achieve maximum repair efficiency. These results show that the catalytic state FADH the sample was purged with high-purity photocycle of DNA repair by photolyase is through a radical nitrogen to remove onygen and then illuminated with a high- mechanism and completed subnanosecond time scale at the intensity lamp (150 W) under anacrobic coaditions, with a cutoff dynamic active site, with no net change in the redox state of the filter at 550 nm to ensure that the sample flavin cofactor. avelengths of 550 The resulting fully reduced enzyme does not absorb significantly at wavelengths of 500 nm. Sub photocycle Iradicalmechaniumlultrafantkinetia strates of dinucleotide cyclobutane thymine dimer and dimer- containing oligo(dTO and poly dT) were prepared by ace- ne of the detrimental effects of UV radiation on the one-sensitized imadiation with a UVB lamp (8 W) according standard procedures (l7. The mixture of the tally reduced is the formation of cyclobut ane pyrimidine dimers (Prk Pyr) between two adjacent thymine bases in with substrates was prepared under yellow light and DNA. Pyr dimers bring both DNA and RNA polymerases to anacrobic conditions, and it had no measurable absorption standstill and may result in mutation oa cell death. Photoly >500 am. The equilibrium binding constants of E col photol- yase for T

Explanation / Answer

Observing the DNA photolase activity in repairing thymine-thymine dimer in DNA

The photoreactivating enzymes binds to the DNA the in region of the dimer to form a complex that absorb the visible light and catalyses cleavage of the covalent linkage between the compound of the dimer. Photoreaction can occur both in bacteria and mammalian cell.

In the damaged DNA the enzyme photolyase use the light energy to split the uv-insduced cyclobutane dimers. The pyrimidine (Thymine) dimers in an affected DNA vrings the DNA and RNA polymerase to a stand still. The enzyme Photolyase is a flavoprotein and contains two noncovalently bound chromophores of which on chromophore is the fully reduced flavin–adenine dinucleotide (FADH) The catalytic cofactor that carries out the repair function upon excitation by either direct photon absorption or resonance energy transfer from the second chromophore is an antenna pigment that harvests sunlight and enhances repair efficiency. The model for the catalytic reaction (1, 2) proposes that the excited flavin cofactor transfers an electron to the Pyr=Pyr dimer to generate a charge-separated radical pair (FADH• PyrPyr•). The anion ring of the cylco version and the excess electron return to the FADH- so that it can be regenerated to form FADH.

For the inevestigation E.Coli DNA photolyase was used and as the solvation strongly modulates the charge-separation, ring-splitting, and electron-return processes. Resulting in slow charge separation (170 ps) and a stretched-single-exponential-decay dynamics ( 0.71). The charge recombination must be slower than the complete ring splitting (560 ps) to eliminate possible ring reclosure and achieve a maximum-repair quantum yield (0.87).

  Active-site solvation strongly modulates the charge-separation, ring-splitting, and electron-return processes, resulting in slow charge separation (170 ps) and a stretched-single-exponential-decay dynamics ( 0.71). The charge recombination must be slower than the complete ring splitting (560 ps) to eliminate possible ring reclosure and achieve a maximum-repair quantum yield (0.87).

   With femtosecond resolution, we followed the photocycle and mapped out the temporal evolution of catalytic reactions. We captured the catalytic intermediate of flavin radical (FADH•). Active-site solvation was observed to occur on picosecond-to-nanosecond time scales and to have a critical role in the continuous modulation of catalytic reactions. These synergistic motions in the active site of the damaged DNA–enzyme complex, optimized by evolution, reveal the perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency (0.87) on the ultrafast time scale of 560 ps.

REFERENCE:-

1). Sancar, A. (2003) Chem. Rev. 103, 2203–2237.

2.) Park, H. W., Kim, S. T., Sancar, A. & Deisenhofer, J. (1995) Science 268, 1866–1872.

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