Biomedical subjects

L C Kroutil

Publications and source records attributed to L C Kroutil.

3 recordsLinked to original sources

PubMed1999

Deletion errors generated during replication of CAG repeats.

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.

Base Sequence↗

PubMed1998

Effect of accessory proteins on T4 DNA polymerase replication fidelity.

The influence of replication accessory proteins on the fidelity of T4 DNA polymerase has been examined. Steady-state kinetic measurements showed that exonuclease-deficient T4 DNA polymerase, alone or with clamp loaders gp44/gp62 and polymerase clamp gp45, displays decreased binding affinity for incorrect as compared to correct dNTPs and a deceased kcat for misinsertion as compared to correct insertion. Kinetic constants were similar with and without accessory proteins, indicating that accessory proteins had little effect on misinsertion. They also had little effect on the Km value for extension of a T.T mismatch. However, the kcat value for T.T mismatch extension was fivefold higher in the presence of the clamp loader and clamp proteins. Thus, in the absence of proofreading, these accessory proteins may promote stable misincorporation. The kinetic analysis is supported by error rate determinations during gap-filling synthesis, which require both misinsertion and mispair extension. For some mispairs, the accuracy of exonuclease-deficient polymerase alone is similar to that in the presence of clamp loader, clamp and single-stranded DNA binding protein (gp32). However, exonuclease-deficient holoenzyme complex is actually less accurate than the polymerase alone for some base substitutions. We suggest that gp45 promotes extension of mismatches by tethering the polymerase to DNA, a process that may be relevant to replication past lesions or other blocks to DNA synthesis. The error rate for one-nucleotide deletions in homopolymeric runs was similar for the polymerase with or without its accessory proteins. This implies that strand misalignment errors arise during highly processive replication. Thus, either unpaired bases can migrate through the run while the DNA polymerase is bound to the template-primer, or the DNA polymerase dissociates from the DNA to allow misalignment but remains tethered to the template through interactions with the clamp. Finally, the T4 replication accessory proteins reduced by >/=10-fold the rate at which exonuclease-deficient T4 DNA polymerase generated deletions of larger numbers of nucleotides, indicating that these proteins influence replication fidelity for other than single base mutations.

Bacteriophage T4↗

PubMed1996

Exonucleolytic proofreading during replication of repetitive DNA.

We are attempting to understand the processes required to accurately replicate the repetitive DNA sequences whose instability is associated with several human diseases. Here we test the hypothesis that the contribution of exonucleolytic proofreading to frameshift fidelity during replication of repetitive DNA sequences diminishes as the number of repeats in the sequence increases. The error rates of proofreading-proficient T7, T4, and Pyrococcus furiosis DNA polymerases are compared to their exonuclease-deficient derivatives, for +1 and -1 base errors in homopolymeric repeat sequences of three to eight base pairs. All three exonuclease-deficient polymerases produce frameshift errors during synthesis at rates that increase as a function of run length, suggesting the involvement of misaligned intermediates. Their wild-type counterparts are all much more accurate, suggesting that the majority of the intermediates are corrected by proofreading. However, the contribution of the exonuclease to fidelity decreases substantially as the length of the homopolymeric run increases. For example, the exonuclease enhances the frameshift fidelity of T7 DNA polymerase in a run of three A.T base pairs by 160-fold, similar to its contribution to base substitution fidelity. However, in a run of eight consecutive A.T base pairs, the exonuclease only enhances frameshift fidelity by 7-fold. A similar pattern was observed with T4 and Pfu DNA polymerases. Thus, both polymerase selectivity and exonucleolytic proofreading efficiency are diminished during replication of repetitive sequences. This may place an increased relative burden on post-replication repair processes to reduce rates of addition and deletion mutations in organisms whose genome contains abundant simple repeat DNA sequences.

Base Sequence↗