Sub-nuclear irradiation, in-vivo microscopy and single-molecule imaging to study a DNA Polymerase

SORIA G, MANSILLA SF, BELLUSCIO L, SPERONI J, D’ALESSIO C, ESSERS J, KANAAR R, GOTTIFREDI V

Tucumán, Argentina

Congreso; XLIV Reunión Anual; 2009

SAIB

Sub-nuclear irradiation, in-vivo microscopy and single-molecule imaging to study a DNA Polymerase Soria G1, Mansilla S1, Laura Belluscio1, Speroni J1, Essers J2, Kanaar R2, Gottifredi V1 1 Fundación Leloir - Buenos Aires- Argentina 2  Erasmus Medical Center- Rotterdam- The Netherlands When the DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass.  An example of a classical TLS polymerase is Pol h (eta). The current model implies that Pol h activity is circumscribed to S-phase. Here we perform a systematic characterization of Pol h behaviour after DNA-damage. We show that Pol h is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G1. This observation was confirmed by different sub-nuclear damage strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. By local UV irradiation and alpha particle irradiation we evaluated the potential connection between Pol h recruitment to DNA lesions outside S phase and Homologous recombination repair (HRR) or Nucleotide excision repair (NER). Finally, we employ a single-molecule imaging approach (known as DNA fiber-assay) to determine how Pol h influences the progression of the replication fork. Our data reveals that the re-localization of the TLS polymerase Pol h to DNA lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.