IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
A failure to downregulate the negative regulator of TLS, p21, upon UV irradiation generates DNA replication defects that trigger apoptosis and genomic instability.
Autor/es:
MANSILLA SF , SORIA G , VALLERGA MB, SPERONI J ,FEDERICO MB, HABIF M, GOTTIFREDI V.
Reunión:
Conferencia; Gordon research Conference. Mutagenesis.A Delicate Balance: Cellular Mutation Pathways in Genetic Stability and Disease.; 2012
Resumen:
The role of p21 upregulation in cell cycle withdrawal upon genotoxins delivery was carefully characterized. However, it is unclear if endogenous p21 affects the transit though S phase of cycling cells, specially when damaged-DNA accumulates abruptly (e.g.: upon UV irradiation). In this scenario, p21 might not benefit (and moreover, might even hamper) one or more aspects of DDR (DNA damage response). Interestingly, UV light triggers p21 degradation thus emphasizing a potential negative role of p21 in the cellular response to UV-induced DNA damage. Since UV irradiation does not trigger cell cycle arrest we though to explore the role of p21 on damaged DNA replication. Using the DNA fiber assay we monitored single replication forks and found that endogenous p21 impaired fork progression upon DNA damage when samples were compared to p21-/- isogenic controls. Interestingly, by using a stable p21 mutant (sp21) that allows cell cycle progression (does not bind CKDs) we were able to conclude that the negative regulation of fork progression upon damaged DNA accumulation was associated to its PCNA (Proliferating Cell Nuclear Antigen) binding domain. Moreover, DDR markers (focal organization/intensity) such as ?×H2AX and 53BP1 increased when UV-triggered p21 depletion was prevented by the expression of sp21. Cell death and replication-stress associated genomic stability (micronuclei formation) also increased when sp21 was expressed. Importantly, all alterations were no longer observed when the PCNA binding domain of sp21 was disrupted. Since we previously reported a negative role of p21 in the recruitment of specialized pol ?Ø to replication factories we thought that the negative effect of sp21 on damaged DNA replication could result from a global effect of sp21 on translesion DNA synthesis (TLS), an auxiliary replication pathway that facilitates the use of damaged-DNA as replication templates. We observed that sp21 blocked focal organization of Y family polymerases (pol ?Ø, pol ?Ù, pol ?Û and Rev1) upon UV irradiation. Moreover, endogenous p21 also impaired Y family polymerase focal organization during the initial hours upon UV irradiation (which coincided with the timing of p21 by protein degradation). All together our data indicates that the removal of endogenous p21 from cells upon UV irradiation might be necessary to facilitate the up-regulation of TLS events that prevent the cell death and genomic instability associated with excessive replication fork stalling.