The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

TY - JOUR

T1 - The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

AU - Eppink, Berina

AU - Tafel, Agnieszka A

AU - Hanada, Katsuhiro

AU - van Drunen, Ellen

AU - Hickson, Ian D

AU - Essers, Jeroen

AU - Kanaar, Roland

N1 - Copyright © 2011 Elsevier B.V. All rights reserved.

PY - 2011/11/10

Y1 - 2011/11/10

N2 - Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.

AB - Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.

KW - Animals

KW - Cell Survival

KW - Chromosome Aberrations

KW - DNA Breaks, Double-Stranded

KW - DNA Replication

KW - Embryonic Stem Cells

KW - Homologous Recombination

KW - Humans

KW - Mice

KW - Mice, Inbred C57BL

KW - Nuclear Proteins

KW - Proliferating Cell Nuclear Antigen

KW - Protein Transport

KW - Signal Transduction

KW - Ultraviolet Rays

U2 - 10.1016/j.dnarep.2011.08.006

DO - 10.1016/j.dnarep.2011.08.006

M3 - Journal article

C2 - 21885354

SN - 1568-7864

VL - 10

SP - 1095

EP - 1105

JO - DNA Repair

JF - DNA Repair

IS - 11

ER -