Dataset Information

Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions.

ABSTRACT: Polymerases belonging to the DinB class of the Y-family translesion synthesis DNA polymerases have a preference for accurately and efficiently bypassing damaged guanosines. These DinB polymerases also generate single-base (-1) deletions at high frequencies with most occurring on repetitive 'deletion hotspot' sequences. Human DNA polymerase kappa (hPol?), the eukaryotic DinB homologue, displays an unusual efficiency for to extend from mispaired primer termini, either by extending directly from the mispair or by primer-template misalignment. This latter property explains how hPol? creates single-base deletions in non-repetitive sequences, but does not address how deletions occur in repetitive deletion hotspots. Here, we show that hPol? uses a classical Streisinger template-slippage mechanism to generate -1 deletions in repetitive sequences, as do the bacterial and archaeal homologues. After the first nucleotide is added by template slippage, however, hPol? can efficiently realign the primer-template duplex before continuing DNA synthesis. Strand realignment results in a base-substitution mutation, minimizing generation of more deleterious frameshift mutations. On non-repetitive sequences, we find that nucleotide misincorporation is slower if the incoming nucleotide can correctly basepair with the nucleotide immediately 5' to the templating base, thereby competing against the mispairing with the templating base.

SUBMITTER: Mukherjee P

PROVIDER: S-EPMC3643592 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions.

Mukherjee Purba P Lahiri Indrajit I Pata Janice D JD

Nucleic acids research 20130404 9

Polymerases belonging to the DinB class of the Y-family translesion synthesis DNA polymerases have a preference for accurately and efficiently bypassing damaged guanosines. These DinB polymerases also generate single-base (-1) deletions at high frequencies with most occurring on repetitive 'deletion hotspot' sequences. Human DNA polymerase kappa (hPolκ), the eukaryotic DinB homologue, displays an unusual efficiency for to extend from mispaired primer termini, either by extending directly from th ...[more]

PMID: 23558743

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Project description:Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location.IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped.

Dataset Information

Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions.

Publications

Human polymerase kappa uses a template-slippage deletion mechanism, but can realign the slipped strands to favour base substitution mutations over deletions.

Similar Datasets

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