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Human DNA polymerase mu (Pol mu) exhibits an unusual replication slippage ability at AAF lesion
Jean-Baptiste Duvauchelle1, Luis Blanco, Robert P P Fuchs
1UPR9003 du CNRS, Cancérogenèse et Mutagenèse Moléculaire et Structurale, ESBS, Boulevard S. Brant, 67400 Strasbourg, France.
Abstract:
We analyzed the ability of various cell extracts to extend a radiolabeled primer past an N-2-acetylaminofluorene (AAF) adduct located on a primed single-stranded template. When the 3' end of the primer is located opposite the lesion, partially fractionated human primary fibroblast extracts efficiently catalyzed primer-terminus extension by adding a ladder of about 15 dGMPs, in an apparently non-templated reaction. This activity was not detected in SV40-transformed fibroblasts or in HeLa cell extracts unless purified human DNA polymerase mu (Pol mu) was added. In contrast, purified human Pol mu alone could only add three dGMPs as predicted from the sequence of the template. These results suggest that a cofactor(s) present in cellular extracts modifies Pol mu activity. The production of the dGMP ladder at the primer terminus located opposite the AAF adduct reveals an unusual ability of Pol mu (in conjunction with its cofactor) to perform DNA synthesis from a slipped intermediate containing several unpaired bases.
Insights
Cell extracts containing a cofactor enhance DNA polymerase mu activity, enabling it to synthesize DNA past N-2-acetylaminofluorene adducts. This suggests a novel mechanism for DNA repair involving non-templated synthesis from a slipped intermediate.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Enzymology
Background:
- DNA adducts, such as N-2-acetylaminofluorene (AAF), pose significant challenges to DNA replication fidelity.
- Cellular extracts possess factors that can influence DNA polymerase activity and repair processes.
Purpose of the Study:
- To investigate the ability of cell extracts to facilitate primer extension past an AAF adduct.
- To identify the specific DNA polymerase and any cofactors involved in this process.
Main Methods:
- Analysis of primer extension assays using radiolabeled primers and single-stranded DNA templates containing an AAF adduct.
- Fractionation of human primary fibroblast extracts.
- Assays with purified human DNA polymerase mu (Pol mu) alone and in combination with cell extracts.
Main Results:
- Human primary fibroblast extracts catalyzed extensive primer extension (approx. 15 dGMPs) past the AAF adduct in a non-templated manner.
- This activity was absent in SV40-transformed fibroblasts and HeLa cell extracts without added Pol mu.
- Purified Pol mu alone extended the primer by only three dGMPs, indicating a cofactor-mediated enhancement.
- The observed synthesis suggests Pol mu, with its cofactor, can utilize a slipped intermediate with unpaired bases.
Conclusions:
- Cellular extracts contain a cofactor that significantly enhances the activity of human Pol mu.
- This Pol mu-cofactor complex exhibits an unusual ability to synthesize DNA past AAF adducts via a non-templated mechanism, potentially involving slipped intermediates.
- This finding sheds light on novel DNA repair pathways and the versatile functions of DNA polymerases.