12/28/2023 0 Comments A human okazaki fragmentThe 37-nt fragments of the 1-nt DNA and RNA flap-containing substrates and the 36-nt fragments of the control flap-free and G-T substrates were labeled at their 5′ ends with 32P. MutLα endonuclease incises the discontinuous strand four nucleotides downstream from a 1-nt DNA or RNA flap. The data were obtained by quantification of images, including those shown in A, and are presented as averages ± 1 S.D., n ≥3. B, summary of incision of the discontinuous strands of the indicated DNA substrates at sites that are 4-nt 3′ to the flap or control nick. The hybridization probe is complementary to the discontinuous strand. Each diagram also shows the relative position of the hybridization probe ( bar with an asterisk). The diagrams outline the circular DNA substrates. A, representative images showing incision of the discontinuous strands in the presence of MutLα, MutSα, PCNA, RFC, and RPA. The reaction products were separated on alkaline 1.2% agarose gels, transferred onto nylon membranes, hybridized with 32P-labeled oligonucleotide 16, and visualized by phosphorimaging. After a 10-min incubation, the reactions were stopped by the addition of NaOH and EDTA to the final concentrations of 40 and 5 m m, respectively. When MutSα, MutLα, MutLα-E705K, PCNA, RFC, and RPA were present in the reaction mixtures, their concentrations were 40, 16, 16, 24, 4, and 40 n m, respectively. Each DNA incision reaction was carried out in the mixture containing the indicated human proteins and DNA substrate (1.5 n m). The numbers above the bars are the apparent K d values.ġ-nt DNA and RNA flaps activate MutLα endonuclease to incise the discontinuous strands in the presence of MutSα, PCNA, RFC, and RPA. The apparent K d values were calculated using the data that were obtained by quantification of images, including those shown in A. B and C, apparent K d values for binding of yeast MutSα ( B) and human MutSα ( C) to the indicated DNA substrates. Each DNA-binding reaction was carried out in the mixture containing the indicated concentration of yeast MutSα and the indicated DNA substrate (2 n m). A, representative images showing binding of yeast MutSα to the different DNA substrates. Compared with the top strand of the homoduplex or nicked substrate, the top strands of the flapped and 1-nt insertion substrates each contained an extra nucleotide residue, which was necessary to produce the 1-nt flap or 1-nt insertion. The DNA sequences of the homoduplex and nicked DNA substrates were identical to each other and to the his7-2 sequence, in which the majority of +1 frameshifts are formed. The gel mobility shift assays with the oligonucleotide-based DNA substrates and calculations of the apparent K d values were performed as described under “Experimental Procedures.” All six substrates had the same bottom strand. Human and yeast MutSα proteins recognize 1-nt DNA flaps. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. These results suggest that the MMR system contributes to the removal of 1-nucleotide Okazaki fragment flaps.ĭNA endonuclease DNA mismatch repair DNA replication Okazaki fragment maturation cancer genomic instability mutL homolog 1 (MLH1). ATPase and endonuclease mutants of MutLα are defective in the flap removal. In reconstituted human systems, MutSα, proliferating cell nuclear antigen, and replication factor C activate MutLα endonuclease to remove the flaps. In addition, we determined that purified yeast and human MutSα proteins recognize 1-nucleotide DNA and RNA flaps. We found that in the yeast Saccharomyces cerevisiae, the MMR system and the flap endonuclease Rad27 act in overlapping pathways that protect the nuclear genome from 1-bp insertions. In this work, we investigated whether the MMR system is involved in Okazaki fragment maturation. The DNA mismatch repair (MMR) system plays a major role in promoting genome stability and suppressing carcinogenesis.
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