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Rapid and efficient site-specific mutagenesis without phenotypic selection.
- T. Kunkel
- BiologyProceedings of the National Academy of Sciences…
The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine, which is applied to mutations introduced via both oligonucleotides and error-prone polymerization.
Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.
The results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.
DNA Replication Fidelity*
- T. Kunkel
- BiologyJournal of Biological Chemistry
- 23 April 2004
Current understanding of replication fidelity is reviewed, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity.
Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases
- S. A. Nick McElhinny, Brian E Watts, T. Kunkel
- BiologyProceedings of the National Academy of Sciences
- 1 March 2010
Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36- to 190-fold molar excess over their corresponding dNTBs, consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast.
Genome instability due to ribonucleotide incorporation into DNA
The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair, and that defective repair results in replicative stress and genome instability via DNA strand misalignment.
DNA mismatch repair.
This review focuses on the biochemical mechanism of MMR replication errors, on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.
Requirement for PCNA in DNA Mismatch Repair at a Step Preceding DNA Resynthesis
Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.
The fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus is determined and it is demonstrated that the purified polymerase lacks detectable exonucleolytic proofreading activity.
A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair.
Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation.
The hypothesis that mutations in both alleles of the hMLH1 gene are necessary for the manifestation of defective mismatch repair and microsatellite instability and for enhanced MNNG tolerance is supported.