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Replication of ultraviolet-damaged DNA in human cells: different mechanisms for different lesions?

Grant number: 15/00198-0
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2015
Effective date (End): December 31, 2016
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Carlos Frederico Martins Menck
Grantee:Davi Jardim Martins
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:14/15982-6 - Consequences of repair deficiencies in damaged genome, AP.TEM

Abstract

Ultraviolet light (UV) is a highly carcinogenic agent to which all living beings are exposed. When absorbed by the DNA molecule, UV-light induces mainly lesions of two types: cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone (6-4PP). These lesions distort the double helix, promoting a block to the replication and transcription of DNA, what may result in genomic instability and cell death. The nucleotide excision repair (NER) mechanism is responsible for the removal of these DNA damage, but when not removed they may be bypassed by a process known as translesion synthesis (TLS). TLS is, thus, a mechanism of damage tolerance performed by specific DNA polymerases (Pol). There are two proposed models that attempt to describe the TLS mechanism: direct bypass at the blocked replication fork and the gap-filling model. In both cases, it may result in the formation of regions of single stranded DNA (ssDNA), at the template strand, or gaps. In this context, the aim of this project is to determine the contribution of each type of dimer (CPD and 6-4PP) in the formation of ssDNA after irradiation with low doses of UV light. We will employ human NER deficient (XP-C) cell lines also RNAi silenced for TLS Pols such as Pol ·, Pol ¶ and Rev1, using a highly sensitive methodology, based on modified comet assay, to detect the presence of ssDNA. The use of adenovirus vectors for the transduction and expression of specific photolyases will allow investigating the contribution of each type of lesion for gap formation on DNA, during the replicative process.