Asha Mary Joseph,1 Saheli Daw,1 Ismath Sadhir,1,2 and Anjana Badrinarayanan1
1National Centre for Biological Sciences - Tata Institute of Fundamental Research, Bangalore, India
2Max Planck Institute for Terrestrial Microbiology, LOEWE Centre for Synthetic Microbiology (SYNMIKRO), Marburg, Germany
Microbiology, Time-Lapse Microscopy
Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.
Microscopy was performed on a wide-field epifluorescence microscope (Eclipse Ti-2E, Nikon) with a 63X oil immersion objective (plan apochromat objective with NA 1.41) and illumination from pE4000 light source (CoolLED).
Product Associated Features
Each of the four LED channels of the pE-4000 offer individual irradiance control. In addition to the 256 wavelength combinations, this is a flexible system for time-lapse applications where photobleaching and photodamage can be minimised to enable extended analysis.
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