Perturbed replication induced genome wide or at common fragile sites is differently managed in the absence of WRN.
The Werner syndrome protein (WRN) is a member of the RecQ helicase family. Loss of WRN results in a human disease, the Werner syndrome (WS), characterized by high genomic instability, elevated cancer risk and premature aging. WRN is crucial for the recovery of stalled replication forks and possesses both helicase and exonuclease enzymatic activities of uncertain biological significance. Previous work revealed that WRN promotes formation of MUS81-dependent double strand breaks (DSBs) at HU-induced stalled forks, allowing replication restart at the expense of chromosome stability. Here, using cells expressing the helicase- or exonuclease-dead WRN mutant, we show that both activities of WRN are required to prevent MUS81-dependent breakage after HU-induced replication arrest. Moreover, we provide evidence that, in WS cells, DSBs generated by MUS81 do not require RAD51 activity for their formation. Surprisingly, when replication is specifically perturbed at common fragile sites (CFS) by aphidicolin, WRN limits accumulation of ssDNA gaps and no MUS81-dependent DSBs are detected. However, in both cases, RAD51 is essential to ensure viability of WS cells, although by different mechanisms. Thus, the role of WRN in response to perturbation of replication along CFS is functionally distinct from that carried out at stalled forks genome wide. Our results contribute to unveil two different mechanisms used by the cell to overcome the absence of WRN.
Section of Molecular Epidemiology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Ivana
I
Murfuni
Anita
A
De Santis
Maurizio
M
Federico
Margherita
M
Bignami
Pietro
P
Pichierri
Annapaola
A
Franchitto
D002478:Cells, Cultured; D043283:Chromosome Fragile Sites; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000502:physiology; D004720:Endonucleases / Q000502:physiology; D005090:Exodeoxyribonucleases / Q000502:physiology*; D015894:Genome, Human; D006801:Humans; D051135:Rad51 Recombinase / Q000502:physiology; D053484:RecQ Helicases / Q000502:physiology*; D000071657:Werner Syndrome Helicase
D004268:DNA-Binding Proteins; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C498772:MUS81 protein, human; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase