Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells.
Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase.
Institute of Biomedical Sciences, University of São Paulo, SP, 05508-000, Brazil annabel.quinet@gmail.com.
Annabel
A
Quinet
Institute of Biomedical Sciences, University of São Paulo, SP, 05508-000, Brazil.
Davi Jardim
DJ
Martins
Institute of Biomedical Sciences, University of São Paulo, SP, 05508-000, Brazil.
Alexandre Teixeira
AT
Vessoni
CEA, IMETI, SEPIA, Team Cellular Engineering and Human Syndromes, F-92265 Fontenay-aux-Roses, France.
Denis
D
Biard
CNRS-UMR8200, Université Paris Sud, Institut de Cancérologie Gustave Roussy, 94805 Villejuif, France.
Alain
A
Sarasin
CNRS-UMR8200, Université Paris Sud, Institut de Cancérologie Gustave Roussy, 94805 Villejuif, France.
Anne
A
Stary
Institute of Biomedical Sciences, University of São Paulo, SP, 05508-000, Brazil cfmmenck@usp.br.
Carlos Frederico Martins
CF
Menck
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000378:metabolism; D016196:S Phase / Q000235:genetics; D014161:Transduction, Genetic; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D004255:Deoxyribodipyrimidine Photo-Lyase; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases; C118285:REV3L protein, human; C112338:Rad30 protein