Nucleic Acids Research Advance Access originally published online on December 13, 2007
Nucleic Acids Research 2008 36(2):688-696; doi:10.1093/nar/gkm1089
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Nucleic Acids Research, 2008, Vol. 36, No. 2 688-696
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences
Department of Pathology, Gittlen Cancer Research Foundation, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA
*To whom correspondence should be addressed. Tel: +1 717 531 4065; Fax: +1 717 531 5634; Email: kae4{at}psu.edu
Received October 18, 2007. Revised November 19, 2007. Accepted November 19, 2007.
Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]11 microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol 
) inserts dGMP and dCMP within the [T]11 mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol and polymerase 
-primase within a [T]11 allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.