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Single-molecule study of the effects of temperature, pH, and RNA base on the stepwise enzyme kinetics of 10-23 deoxyribozyme.

ABSTRACT: We investigated how the stepwise enzyme kinetics of 10-23 deoxyribozyme was affected by temperature, pH, and RNA residue of the substrate at the single-molecule level. A deoxyribozyme-substrate system was employed to temporally categorize a single-turnover reaction into four distinct steps: binding, cleavage, dissociation of one of the cleaved fragments, and dissociation of the other fragment. The dwell time of each step was measured as the temperature was varied from 26 to 34 °C, to which the transition state theory was applied to obtain the enthalpy and entropy of activation for individual steps. In addition, we found that only the cleavage step was significantly affected by pH, indicating that it involves deprotonation of a single proton. We also found that different RNA residues specifically affect the cleavage step and cause the dwell time to change by as much as 5 times.

SUBMITTER: Jung J

PROVIDER: S-EPMC9113834 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Single-molecule study of the effects of temperature, pH, and RNA base on the stepwise enzyme kinetics of 10-23 deoxyribozyme.

Jung Jiwon J Kim Seon Yong SY Kim Seong Keun SK

RSC advances 20220518 23

We investigated how the stepwise enzyme kinetics of 10-23 deoxyribozyme was affected by temperature, pH, and RNA residue of the substrate at the single-molecule level. A deoxyribozyme-substrate system was employed to temporally categorize a single-turnover reaction into four distinct steps: binding, cleavage, dissociation of one of the cleaved fragments, and dissociation of the other fragment. The dwell time of each step was measured as the temperature was varied from 26 to 34 °C, to which the t ...[more]

PMID: 35702195

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Project description:The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

Dataset Information

Single-molecule study of the effects of temperature, pH, and RNA base on the stepwise enzyme kinetics of 10-23 deoxyribozyme.

Publications

Single-molecule study of the effects of temperature, pH, and RNA base on the stepwise enzyme kinetics of 10-23 deoxyribozyme.

Similar Datasets

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