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Published on: September 23, 2015
poliota-dependent lesion bypass in vitro
Alexandra Vaisman1, Ekaterina G Frank, John P McDonald
1Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Building 6, Room 1A13, 9000 Rockville Pike, Bethesda, MD 20892-2725, USA.
Abstract:
Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polkappa)-like; Rev1-like, Rad30A (poleta)-like and Rad30B (poliota)-like polymerases. The polIV/polkappa-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and poleta-like polymerases are found exclusively in eukaryotes, and to date, poliota-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the poliota-like polymerases, where the enzymatic properties of Drosophila melanogaster poliota are more similar to that of Saccharomyces cerevisiae and human poleta than to the related human poliota. For example, like poleta, Drosophila poliota can bypass a cis-syn thymine-thymine dimer both accurately and efficiently, while human poliota bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human poliota can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3'T of a 6-4-thymine-thymine pyrimidine-pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although poliota most likely arose from a genetic duplication of poleta millions of years ago as eukaryotes evolved, it would appear that poliota from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have "tailored" its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian poliota plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for poliota and the identification of mice deficient in poliota.
Insights
DNA polymerases in the Y-family have diverse roles. Human poliota, unlike related polymerases, shows reduced DNA lesion bypass efficiency, suggesting specialized functions in higher eukaryotes.
Area of Science:
- Molecular Biology
- Genetics
- Evolutionary Biology
Background:
- The Y-family of DNA polymerases comprises UmuC (polV)-like, DinB (polIV/polkappa)-like, Rev1-like, Rad30A (poleta)-like, and Rad30B (poliota)-like polymerases.
- PolIV/polkappa are ubiquitous, polV are bacterial, Rev1 and poleta are eukaryotic, and poliota are found in higher eukaryotes.
Purpose of the Study:
- To investigate the phylogenetic relationships and in vitro enzymatic properties of Y-family DNA polymerases.
- To compare the lesion bypass capabilities of Drosophila melanogaster poliota, Saccharomyces cerevisiae poleta, and human poliota.
Main Methods:
- Phylogenetic analysis of Y-family DNA polymerases.
- In vitro characterization of DNA polymerase enzymatic properties, focusing on lesion bypass fidelity and efficiency.
Main Results:
- Drosophila poliota efficiently and accurately bypasses cis-syn thymine-thymine dimers, similar to human poleta.
- Human poliota exhibits inefficient and low-fidelity bypass of thymine-thymine dimers.
- While human poliota can insert bases opposite various lesions, subsequent DNA extension is often limited.
Conclusions:
- Human poliota has evolved distinct enzymatic properties compared to poleta, likely adapted for functions beyond DNA lesion bypass in higher eukaryotes.
- Further research using poliota-deficient cell lines and mice is needed to elucidate mammalian poliota's specific functions and in vivo roles in DNA repair.
