PCNA is recruited to irradiated chromatin in late S-phase and is most pronounced in G2 phase of the cell cycle.
DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic. bartova@ibp.cz.
Eva
E
Bártová
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic.
Jana
J
Suchánková
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic.
Soňa
S
Legartová
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic.
Barbora
B
Malyšková
Institute of Cellular Biology and Pathology, the First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 01, Prague, Czech Republic.
Matúš
M
Hornáček
Institute of Cellular Biology and Pathology, the First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 01, Prague, Czech Republic.
Magdalena
M
Skalníková
Institute of Cellular Biology and Pathology, the First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 01, Prague, Czech Republic.
Martin
M
Mašata
Institute of Cellular Biology and Pathology, the First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 01, Prague, Czech Republic.
Ivan
I
Raška
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic.
Stanislav
S
Kozubek
D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology; D016195:G2 Phase / Q000235:genetics / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D016196:S Phase / Q000235:genetics / Q000502:physiology
D002843:Chromatin; D018809:Proliferating Cell Nuclear Antigen
DNA repair; Lamins; Micro-irradiation; PCNA; S/G2 phases; rDNA