2015 CFAI Special Seminar
Confocal microscopy studies of DNA repair processes
in live cells
Inducing low level DNA damage using focused visible light tostudy recruitment and disengagement of repair factors

Dr. Jurek W. Dobrucki
Professor of Jagiellonian University(Krakow, Poland)

Date / Time : September 29th, 2015 / 16:00-17:00: The moderate meeting room (事務棟 中会議室)
Venue
Modern optical microscopy, including fluorescence confocal microscopy and new super-resolution
techniques, open new avenues of research into mechanisms of DNA damage and repair. Microscopy
enables studies of dynamics of recruitment and disengagement of repair factors, changes of chromatin
structure associated with repair processes, choices of the repair pathway and spatial organisation of repair
factors engaged at each damage site, the fate of individual cells following induction of DNA damage, andmany more aspects that cannot be accessed directly by standard biochemical assays that embrace andaverage information from large cell populations.
The precondition for acquiring relevant information regarding mechanisms of DNA repair processes
is an ability to induce a very low number of well defined damage events in a selected region of the nucleus,
or within a selected gene. So far many laboratories relied in their work on inducing multiple foci of DNA
damage by exposing cells to cytotoxic drugs, ionising radiation or UV. Inducing local DNA damage, confinedto very small regions of the nucleus, was achieved by exposing cells to photo-sensitisers combined with UV
or visible light. In most cases, however, several types of DNA damage were induced in one spot (oxidative,DNA strand breaks, etc.) and the damage was too extensive to be repaired, therefore interpretation of theresults of such experiments was complex and cumbersome. Recently a method of inducing individual welldefined damage events, using low intensity visible light in the absence of exogenous photosensitisers, hasbeen proposed [1, 4] and successfully used in studies of a number of repair factors, including HP1, 53BP1and XRCC1 [2,3,4]. I will discuss critical aspects, advantages and limitations of this approach, including theknown aspects of the mechanism of damage induction by visible light, the types of damage induced, andthe role of photon energy and the light dose. I will also present examples of experiments where inductionof individual damage events by low level visible light enabled gaining new information which was difficult toaccess by other, less specific methods of inducing local DNA damage.
1, Solarczyk KJ, Zarębski M, Dobrucki JW. Inducing local DNA damage by visible light to study chromatin repair. DNA
Repair (Amst). 2012 Dec 1;11(12):996-1002.
2, Trembecka-Lucas DO, Szczurek AT, Dobrucki JW. Dynamics of the HP1β-PCNA-containing complexes in DNA
replication and repair. Nucleus. 2013 Jan-Feb;4(1):74-82.
3, Trembecka-Lucas DO, Dobrucki JW. A heterochromatin protein 1 (HP1) dimer and a proliferating cell nuclearantigen (PCNA) protein interact in vivo and are parts of a multiprotein complex involved in DNA replication and DNArepair. Cell Cycle. 2012 Jun 1;11(11):2170-5.
4, Solarczyk K, Kordon M, Dobrucki JW. Two stages of XRCC1 recruitment and two classes of XRCC1 foci formed in
response to low level DNA damage induced by visible light, or stress triggered by heat shock (under revision).
Graduate School of Agricultural Science, TOHOKU UNIVERSITY