To Slip or Skip, Visualizing Frameshift Mutation Dynamics for Error-prone DNA Polymerases

Brigette Tippin,Sawami Kobayashi,Jeffrey G Bertram,Myron F Goodman

doi:10.1074/jbc.m408600200

Brigette Tippin, Sawami Kobayashi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m408600200

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Abstract

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol mu and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol mu switches to dNTP-stabilized misalignment. pol beta generates -1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.

Highlights

Pre-steady state kinetic studies of DNA polymerase fidelity have been focused on base substitution mutagenesis mechanisms [1, 2]
We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo
In this study we have addressed the mechanism by which pol ␮ and pol IV rearrange p/t DNA to cause Ϫ1 frameshifts

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of Native Human pol ␮ and E. coli pol IV—pol ␮ cDNA was subcloned into vector pET41b (Novagen) and expressed in strain BL21(DE3) R1L Codon Plus (Stratagene). Pol ␮-containing fractions were loaded onto a Superdex 200 column (Amersham Biosciences) in PC buffer plus 250 mM NaCl. The cleanest fractions were applied to a Heparin Hi-Trap column (Amersham Biosciences), washed with 20 column volumes of PC buffer supplemented. The off rate (koff) for pol ␮ from rhodamine-labeled p/t DNA was determined using a pre-steady state ⌸*180 stopped-flow instrument from Applied Photophysics equipped for anisotropy by exciting with vertically polarized light at 580 nm (2-nm slit width) and monitoring emissions using a 620 cutoff filter. Steady State 2AP Fluorescence Methodology—Experiments using the non-reiterative p/t and DNA pol ␮ were performed using a QuantaMaster QM-1 fluorometer (Photon Technology International) by exciting 2AP at 310 nm (6-nm band pass) and monitoring the emission at 370 nm (6-nm band pass).

RESULTS

Real Time Deletion Mutation Analysis

Findings

DISCUSSION