TY - JOUR
T1 - UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells
AU - Nishi, Ryotaro
AU - Alekseev, Sergey
AU - Dinant, Christoffel
AU - Hoogstraten, Deborah
AU - Houtsmuller, Adriaan B.
AU - Hoeijmakers, Jan H.J.
AU - Vermeulen, Wim
AU - Hanaoka, Fumio
AU - Sugasawa, Kaoru
N1 - Funding Information:
We thank all lab members in the Biosignal Research Center, Kobe University and at RIKEN for beneficial discussions and encouragement. We also thank all the members in the Erasmus Medical Center, especially S. Bergink, for helpful discussion. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency. This work was also financially supported by the Bioarchitect Research Project of RIKEN. R.N. was supported by the RIKEN Special Postdoctoral Researchers Program.
PY - 2009/6/4
Y1 - 2009/6/4
N2 - Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.
AB - Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.
KW - Dynamics
KW - Nucleotide excision repair
KW - UV-DDB
KW - XPC
UR - http://www.scopus.com/inward/record.url?scp=67349276614&partnerID=8YFLogxK
U2 - 10.1016/j.dnarep.2009.02.004
DO - 10.1016/j.dnarep.2009.02.004
M3 - Article
C2 - 19332393
AN - SCOPUS:67349276614
SN - 1568-7864
VL - 8
SP - 767
EP - 776
JO - DNA Repair
JF - DNA Repair
IS - 6
ER -