SUB-NUCLEAR IRRADIATION, IN-VIVO MICROSCOPY AND SINGLE-MOLECULE IMAGING TO STUDY A DNA POLYMERASE

SORIA G; MANSILLA S; BELLUSCIO L; SPERONI J; D' ALESSIO C; ESSERS J; KANAAR R; GOTTIFREDI V

San MIguel de Tucumán, Tucumán, Argentina

Congreso; 45 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

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 Pol h to DNA lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.