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'External' proofreading of DNA replication errors and mammalian autonomous 3'-->5'exonucleases
I V Shevelev1, T P Kravetskaya, O K Legina
1Department of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute of the Russia Academy of Sciences, Gatchina, Leningrad District, Russia.
Abstract:
Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. The base substitutions misinserted by these polymerases could be eliminated with two kinds of an 'external' proofreading carried out (1) by the 3'-->5' exonuclease function intrinsic to DNA polymerases delta and epsilon or/and (2) by the autonomous 3'-->5' exonucleases non-associated covalently with DNA polymerases. DNA polymerases delta and epsilon can be separated from autonomous 3'-->5' exonucleases by means of sedimentation. Ultracentrifugation of the nuclear extracts and cytosols from normal and regenerating rat liver as well as from total embryos has shown the bulk of the cellular 3'-->5' exonucleolytic activity is due to autonomous nucleases. Moreover, the level of such a specific activity correlates with the replicative status of the organs from adult animals: spleen > regenerating liver > normal liver > cardiac muscle > brain, maximum difference being an order of magnitude. In addition, autonomous exonucleases were shown to be the constituents of the multienzyme forms of DNA polymerases alpha and beta. Hence, autonomous 3'-->5' exonucleases seem to be the principal participants in an 'external' proofreading.
Insights
Mammalian DNA polymerases alpha and beta lack proofreading. Autonomous 3'-->5' exonucleases, found in various rat tissues, likely provide essential external proofreading to correct DNA replication errors.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Mammalian nuclear DNA polymerases alpha and beta lack intrinsic 3'-->5' exonucleolytic proofreading activity.
- DNA base substitutions by these polymerases require external mechanisms for correction.
- Proofreading can be mediated by DNA polymerases delta/epsilon or autonomous exonucleases.
Purpose of the Study:
- To investigate the role and prevalence of autonomous 3'-->5' exonucleases in DNA repair.
- To determine the contribution of autonomous exonucleases to the overall proofreading activity in mammalian cells.
Main Methods:
- Ultracentrifugation of nuclear extracts and cytosols from rat tissues (liver, spleen, brain, heart) and embryos.
- Separation of DNA polymerases delta and epsilon from autonomous 3'-->5' exonucleases.
- Analysis of 3'-->5' exonuclease activity levels in correlation with organ replicative status.
Main Results:
- The majority of cellular 3'-->5' exonucleolytic activity originates from autonomous nucleases, not polymerases delta/epsilon.
- Exonuclease activity levels correlate with tissue replicative rates (spleen > regenerating liver > normal liver > cardiac muscle > brain).
- Autonomous exonucleases are components of DNA polymerases alpha and beta multi-enzyme complexes.
Conclusions:
- Autonomous 3'-->5' exonucleases are the primary mediators of external proofreading in mammalian cells.
- These exonucleases play a crucial role in maintaining genomic integrity by correcting replication errors.
- Their association with DNA polymerases alpha and beta suggests a direct role in DNA synthesis fidelity.